Coupling method

ABSTRACT

The invention relates to a new method of determining the presence, absence or characteristics of an analyte. The analyte is coupled to a membrane. The invention also relates to nucleic acid sequencing.

RELATED APPLICATIONS

This application is a 35 U.S.C. 371 national stage filing ofInternational Application No. PCT/GB2012/051191 filed May 25, 2012,which claims priority to U.S. Application Nos. 61/490,860 filed May 27,2011; and 61/599,246 filed Feb. 15, 2012. The contents of theaforementioned applications are hereby incorporated by reference.

FIELD OF THE INVENTION

The invention relates to a new method of determining the presence,absence or characteristics of an analyte. The analyte is coupled to amembrane. The invention also relates to nucleic acid sequencing.

BACKGROUND OF THE INVENTION

There is currently a need for rapid and cheap nucleic acid (e.g. DNA orRNA) sequencing technologies across a wide range of applications.Existing technologies are slow and expensive mainly because they rely onamplification techniques to produce large volumes of nucleic acid andrequire a high quantity of specialist fluorescent chemicals for signaldetection.

Nanopores have great potential as direct, electrical biosensors forpolymers and a variety of small molecules. In particular, recent focushas been given to nanopores as a potential DNA sequencing technology.Two methods for DNA sequencing have been proposed; ‘ExonucleaseSequencing’, where bases are processively cleaved from thepolynucleotide by an exonuclease and are then individually identified bythe nanopore and also ‘Strand Sequencing’, where a single DNA strand ispassed through the pore and nucleotides are directly identified. StrandSequencing may involve the use of a DNA handling enzyme to control themovement of the polynucleotide through the nanopore.

When a potential is applied across a nanopore, there is a drop in thecurrent flow when an analyte, such as a nucleotide, resides transientlyin the barrel for a certain period of time. Nanopore detection of theanalyte gives a current blockade of known signature and duration. Theconcentration of an analyte can then be determined by the number ofblockade events per unit time to a single pore.

For nanopore applications, such as DNA Sequencing, efficient capture ofanalyte from solution is required. For instance, in order to give theDNA handling enzyme used in DNA Sequencing a sufficiently high dutycycle to obtain efficient sequencing, the number of interactions betweenenzyme and polynucleotide needs to be maximal, so that a newpolynucleotide is bound as soon as the present one is finished.Therefore, in DNA Sequencing, it is preferred to have the polynucleotideat as high a concentration as is possible so that, as soon as an enzymefinishes processing one, the next is readily available to be bound. Thisbecomes a particular problem as the concentration of polynucleotide,such as DNA, becomes limiting, e.g. DNA from cancer cell samples forepigenetics. The more dilute the sample then the longer betweensequencing runs, up to the point where binding the first polynucleotideis so limiting that it is unfeasible.

The limits of nanopore detection have been estimated for variousanalytes. Capture of a 92-nucleotide synthetic piece of single strandDNA (ssDNA) by a protein nanopore (hemolysin) was determined to be at afrequency of 3.0±0.2 s⁻¹ uM⁻¹ (Maglia, Restrepo et al. 2008, Proc NatlAcad Sci USA 105(50): 19720-5). Capture could be increased ˜10 fold bythe addition of a ring of positive charges at the entrance to thehemolysin barrel (23.0±2 s⁻¹ uM⁻¹). To put this into context, 1 uM of 92nucleotide ssDNA is equivalent to 31 ug of DNA required per singlechannel recording, assuming a cis chamber volume of 1 ml. The marketleading genomic DNA purification kit from human blood (Qiagen's PAXgeneBlood DNA Kit) currently gives expected yields of between 150-500 ug ofgenomic from 8.5 ml of human whole blood. Therefore, this disclosedincrease in analyte detection is still well short of the step changerequired for ultra-sensitive detection and delivery.

SUMMARY OF THE INVENTION

The inventors have surprisingly demonstrated ultra low concentrationanalyte delivery by coupling the analyte to a membrane in which therelevant detector is present. This lowers by several orders of magnitudethe amount of analyte required in order to be detected. The extent towhich the amount of analyte needed is reduced could not have beenpredicted.

In particular, the inventors surprisingly report an increase in thecapture of single stranded DNA by ˜4 orders of magnitude over thatpreviously reported. As both the detector and analyte are now on thesame plane, then ˜10³ M s⁻¹ more interactions occur per second, asdiffusion of both molecules is in two dimensions rather than threedimensions. This has dramatic implications on the sample preparationrequirements that are of key concern for diagnostic devices such asnext-generation sequencing systems.

In addition, coupling the analyte to a membrane has added advantages forvarious nanopore-enzyme sequencing applications. In ExonucleaseSequencing, when the DNA analyte is introduced the pore may becomeblocked permanently or temporarily, preventing the detection ofindividual nucleotides. When one end of the DNA analyte is localisedaway from the pore, for example by coupling or tethering to themembrane, surprisingly it was found that this temporary or permanentblocking is no longer observed. By occupying one end of the DNA bycoupling it to the membrane it also acts to effectively increase theanalyte concentration over the detector and so increase the sequencingsystems duty cycle. This is discussed in more detail below.

Accordingly, the invention provides a method for determining thepresence, absence or characteristics of an analyte, comprising (a)coupling the analyte to a membrane and (b) allowing the analyte tointeract with a detector present in the membrane and thereby determiningthe presence, absence or characteristics of the analyte.

The invention also provides:

-   -   a method of sequencing an analyte which is a target        polynucleotide, comprising:        -   (a) coupling the target polynucleotide to a membrane;        -   (b) allowing the target polynucleotide to interact with a            detector present in the membrane, wherein the detector            comprises a transmembrane pore and an exonuclease, such that            the exonuclease digests an individual nucleotide from one            end of the target polynucleotide;        -   (c) allowing the nucleotide to interact with the pore;        -   (d) measuring the current passing through the pore during            the interaction and thereby determining the identity of the            nucleotide; and        -   (e) repeating steps (b) to (d) at the same end of the target            polynucleotide and thereby determining the sequence of the            target polynucleotide;    -   a method of sequencing an analyte which is a target        polynucleotide, comprising:        -   (a) coupling the target polynucleotide to a membrane;        -   (b) allowing the target polynucleotide to interact with a            detector present in the membrane, wherein the detector            comprises a transmembrane pore, such that the target            polynucleotide moves through the pore; and        -   (c) measuring the current passing through the pore as the            target polynucleotide moves with respect to the pore and            thereby determining the sequence of the target            polynucleotide;    -   a kit for sequencing an analyte which is a target polynucleotide        comprising (a) a transmembrane pore, (b) a polynucleotide        binding protein and (c) means to couple the target        polynucleotide to a membrane; and    -   an apparatus for sequencing an analyte which is a target        polynucleotide, comprising (a) a membrane, (b) a plurality of        transmembrane pores in the membrane, (c) a plurality of        polynucleotide binding proteins and (d) a plurality of target        polynucleotides coupled to the membrane.

DESCRIPTION OF THE FIGURES

FIG. 1 shows nanopore sensing of an analyte. A) Shows a nanopore withthe direction of the current flow indicated by grey arrows. A predictedcurrent trace is shown below. B) Shows a nanopore with an analytetranslocating through the pore. The direction of analyte movement isindicated by arrow 1 and the direction of the current flow by the greyarrows. A predicted current trace is shown below showing how the currentchanges as the analyte translocates through the pore.

FIG. 2 shows a method for tethering DNA nanopore interactions. SectionsA and B show transient tethered ssDNA and how the current trace changesas the ssDNA translocates through the pore. Sections C and D show stabletethered ssDNA and how the current trace changes as the ssDNA iscaptured by the pore.

FIG. 3 shows capture of a DNA-enzyme complex, followed by dissociationof the DNA and the enzyme, and subsequent DNA de-hybridisation.

FIG. 4 shows the experimental setup for Example 2. Comparison between(1) a primer/template DNA analyte in solution (A—top) where theconcentrations of material are in the high nanomolar range (400 nM DNAused and 800 nN enzyme used) and (2) a tethered system (B—bottom) wherethe amount of material is sub-nanomolar (1 nM DNA used and 5 nN enzymeused).

FIG. 5 shows KF binding times on top of the nanopore for non-tetheredanalyte (DNA) in the absence of KF (DNA concentration=400 nM).

FIG. 6 shows KF binding times on top of the nanopore for non-tetheredanalyte (DNA) in the presence of KF (DNA concentration=400 nM, KFconcentration=800 nM). KF binding was 1-100 ms.

FIG. 7 shows KF binding times on the top of the nanopore for tetheredanalyte (DNA) in the absence of KF (DNA concentration=1 nM).

FIG. 8 shows KF binding times on top of the nanopore for tetheredanalyte (DNA) in the presence of KF (DNA concentration=1 nM, KFconcentration=5 nM). KF binding was 0.1-10 s.

FIG. 9 shows an example of a Phi29 DNA polymerase mediated unzippingevent of transiently tethered dsDNA. The drop in current from the openpore level is thought to be a blockade caused by capturing a DNA:proteincomplex. This captured complex resides on the nanopore for ˜5 secondsgiving a constant current level before rapidly changing between levelsand then finally returning to the open pore level. This is thought to bea pause before unzipping is initiated and a single A moves through thereader head so giving the oscillation in current. When the duplex hasbeen fully unzipped the target strand translocates, the primer andpolymerase dissociate and so the current returns to the open pore level.

FIG. 10 shows an example of a Phi29 DNA polymerase mediated unzippingevent of solution dsDNA. The drop in current from the open pore level isthought to be a blockade caused by capturing a DNA:protein complex. Thiscaptured complex resides on the nanopore for ˜12 seconds giving aconstant current level before rapidly changing between levels and thenfinally returning to the open pore level. This is thought to be a pausebefore unzipping is initiated and as the single A moves through thereader head so giving the oscillation in current. When the duplex hasbeen fully unzipped the target strand translocates, the primer andpolymerase dissociate and so the current returns to the open pore level.

FIG. 11 shows an example of event sequences from one unzipping run fornon-tethered dsDNA analyte. The number of levels observed as well as thelevel and duration for these are broadly consistent with the tetheredexperiments.

FIG. 12 shows an example of event sequences from one unzipping run fortethered dsDNA analyte. The number of levels observed as well as thelevel and duration for these are broadly consistent with the solution(non-tethered) DNA experiments.

FIG. 13 shows a plasmid map of tethered strand sequencing analytes fromgenomic DNA. Primers were designed complementary to PhiX 174 genomicDNA. The same sense primer was used for all and contained a 5′-50polyTregion followed by 4 abasic sites before the complementary region. Thehybridisation sites for the antisense primers were varied according tothe desired fragment size. Each antisense primer contained a5′-cholesterol group.

FIG. 14 shows PCR generation of tethered strand sequencing analytes fromgenomic DNA. Primers were designed complementary to PhiX 174 genomicDNA. The same sense primer was used for all and contained a 5′-50polyTregion followed by 4 abasic sites before the complementary region. Thehybridisation sites for the antisense primers were varied according tothe desired fragment size. Each antisense primer contained a5′-cholesterol group. To confirm presence of the 50polyT region to the5′ of the sense strand, fragments were digested with the 5′-3′ singlestrand specific RecJ exonuclease (NEB) and this was analysed on a gel.Lane 1 contains 50 nt ssDNA, 235 bp dsDNA only. Lane 2 contains 50 ntssDNA, 235 bp dsDNA which has been digested with the 5′-3′ single strandspecific RecJ exonuclease (NEB). Lane 3 contains 50 nt ssDNA, 400 bpdsDNA only. Lane 4 contains 50 nt ssDNA, 400 bp dsDNA which has beendigested with the 5′-3′ single strand specific RecJ exonuclease (NEB).Lane 5 contains 50 nt ssDNA, 835 bp dsDNA only. Lane 6 contains 50 ntssDNA, 835 bp dsDNA which has been digested with the 5′-3′ single strandspecific RecJ exonuclease (NEB).

FIG. 15 shows unzipping events from the 800 bp PhiX 174 amplifiedfragment. This 800 bp sequence corresponds to the sequence betweenpoints 1 and 3 in the plasmid map shown.

FIG. 16 shows unzipping events from the 200 bp PhiX 174 amplifiedfragment. This 200 bp sequence corresponds to the sequence betweenpoints 1 and 2 in the plasmid map shown. The 200mer is aligned againstthe 800mer sequences shown in FIG. 15 with zero leading and trailing gappenalties (i.e. it is free to start anywhere, but “internal” gaps arepenalised). As expected, the 200mer sections align with the front of the800mer.

FIG. 17 shows analyte tethering schemes for solid state nanopores. A)Shows tethering into a modified surface (tethering in a layer). B) Showstethering to a modified surface (interaction with the surface). C) Showstethering to a lipid monolayer on a modified surface. D) Shows tetheringto a lipid bilayer on a modified surface.

FIG. 18 shows methods for coupling double stranded polynucleotides to alipid membrane. A) Shows a single tethered dsDNA binding proteininteracting with dsDNA analyte. B) Shows multiple tethered dsDNA bindingproteins interacting with a single dsDNA analyte. C) Shows a singletethered chemical group interacting with dsDNA analyte.

FIG. 19 shows methods for coupling single stranded polynucleotideanalytes to lipid membranes. A) Shows a single tethered ssDNA bindingprotein interacting with ssDNA. B) Shows multiple tethered ssDNA bindingproteins interacting with a single ssDNA. C) Shows a single tetheredchemical group interacting with ssDNA.

FIG. 20 shows a schematic of one way of using a polynucleotide bindingprotein to control DNA movement through a nanopore employing a dsDNAbinding protein to couple the DNA to the membrane. A) A DNA analyte(consisting of a ssDNA leader (grey region) attached to a dsDNA region)is coupled to the membrane using a tethered dsDNA binding protein,resulting in a concentration enhancement at the membrane surface. Apolynucleotide binding protein capable of controlling polynucleotidemovement is added to the cis compartment where it binds to the 4 bpoverhang B) Under an applied voltage, the DNA analyte is captured by thenanopore via the 5′ leader section (grey region) on the DNA. C) Underthe force of the applied field the DNA is pulled into the pore until thebound polynucleotide binding protein contacts the top of the pore andprevents further uncontrolled translocation. In this process theantisense strand is stripped from the DNA strand, therefore, resultingin the detachment of the dsDNA binding protein from the strand. D) Inthe presence of appropriate cofactors, the polynucleotide bindingprotein on top of the pore moves along the DNA and controls thetranslocation of the DNA through the pore. The movement of thepolynucleotide binding protein, along the DNA in a 3′ to 5′ direction,pulls the threaded DNA out of the pore against the applied field back tothe cis compartment. The last section of DNA to pass through thenanopore is the 5′-leader. The arrow indicates the direction of DNAmovement.

FIG. 21 shows a schematic of one way of using a polynucleotide bindingprotein to control DNA movement through a nanopore employing ahybridised tether. A) A DNA analyte (consisting of a ssDNA leader (greyregion) attached to a dsDNA region) is coupled to the membrane using ahybridised tether, resulting in a concentration enhancement at themembrane surface. A polynucleotide binding protein capable ofcontrolling DNA movement is added to the cis compartment where it bindsto the 4 bp overhang. B) Under an applied voltage, the DNA analyte iscaptured by the nanopore via the 5′ leader section (grey region) on theDNA. C) Under the force of the applied field the DNA is pulled into thepore until the bound polynucleotide binding protein contacts the top ofthe pore and prevents further uncontrolled translocation. In thisprocess the polynucleotide which is tethered to the membrane (dashedline) is stripped off to be sequenced (black strand with grey leaderregion). D) In the presence of appropriate cofactors, the polynucleotidebinding protein on top of the pore moves along the DNA and controls thetranslocation of the DNA through the pore. The movement of thepolynucleotide binding protein, along the DNA in a 3′ to 5′ direction,pulls the threaded DNA out of the pore against the applied field back tothe cis compartment. The last section of DNA to pass through thenanopore is the 5′-leader. The arrow indicates the direction of DNAmovement.

FIG. 22 shows a schematic of one way of using a polynucleotide bindingprotein to control DNA movement through a nanopore employing ahybridised tether. A) A DNA analyte (consisting of ssDNA (black linewith the leader sequence shown in grey) hybridised to a ssDNA tether(dashed line)) is coupled to the membrane using a hybridised tether,resulting in a concentration enhancement at the membrane surface. Apolynucleotide binding protein capable of controlling DNA movement isadded to the cis compartment where it binds to the 4 bp overhang. B)Under an applied voltage, the DNA analyte is captured by the nanoporevia the 5′ leader section (grey region) on the DNA. C) Under the forceof the applied field the DNA is pulled into the pore until the boundpolynucleotide binding protein contacts the top of the pore and preventsfurther uncontrolled translocation. In this process the strand which istethered to the membrane (dashed line) is stripped off the ssDNA strandto be sequenced (black strand with grey leader region). D) In thepresence of appropriate cofactors, the polynucleotide binding protein ontop of the pore moves along the DNA and controls the translocation ofthe DNA through the pore. The movement of the polynucleotide bindingprotein, along the DNA in a 3′ to 5′ direction, pulls the threaded DNAout of the pore against the applied field back to the cis compartment.The last section of DNA to pass through the nanopore is the 5′-leader.The arrow indicates the direction of DNA movement.

FIG. 23 shows several methods of tethering a probe, which can beemployed for the detection of microRNA, to a membrane. A) The probe canbe permanently tethered to the membrane. In this instance the region ofthe probe that hybridises to the microRNA is in the middle of the probe.The barcoded region (dotted region) of the probe, which is used toidentify the probe, is located at the opposite end of the strand to thetether. Bi and ii) The probe can be transiently tethered to the membraneby internal hybridisation. In this example the region of the probe thathybridises to the microRNA is attached to one end of the strand. Thebarcoding region (dotted region), which is used to identify the probe,is located directly above the tether and below the microRNAhybridisation region. In Bii) the hybridisation region of the tether tothe probe is inverted in its binding direction in comparison to Bi). Ciand ii) The probe can be transiently tethered to the membrane byhybridisation to one end of the probe. In this example the region of theprobe that hybridises to the microRNA is located in the middle of thestrand. The barcoding region (dotted region), which is used to detectthe presence or absence of the microRNA, is located below the microRNAhybridisation region at the opposite end of the probe to the tether. InCii) the hybridisation region of the tether to the probe is inverted inits binding direction in comparison to Ci).

DESCRIPTION OF THE SEQUENCE LISTING

SEQ ID NO: 1 shows the codon optimised polynucleotide sequence encodingthe NNN-RRK mutant MspA monomer.

SEQ ID NO: 2 (also referred to as “B1”) shows the amino acid sequence ofthe mature form of the NNN-RRK mutant of the MspA monomer. The mutantlacks the signal sequence and includes the following mutations: D90N,D91N, D93N, D118R, D134R and E139K. These mutations allow DNA transitionthrough the MspA pore.

SEQ ID NO: 3 shows the polynucleotide sequence encoding one subunit ofα-hemolysin-M111R (α-HL-R).

SEQ ID NO: 4 shows the amino acid sequence of one subunit of α-HL-R.

SEQ ID NO: 5 shows the codon optimised polynucleotide sequence encodingthe Phi29 DNA polymerase.

SEQ ID NO: 6 shows the amino acid sequence of the Phi29 DNA polymerase.

SEQ ID NO: 7 shows the codon optimised polynucleotide sequence derivedfrom the sbcB gene from E. coli. It encodes the exonuclease I enzyme(EcoExo I) from E. coli.

SEQ ID NO: 8 shows the amino acid sequence of exonuclease I enzyme(EcoExo I) from E. coli.

SEQ ID NO: 9 shows the codon optimised polynucleotide sequence derivedfrom the xthA gene from E. coli. It encodes the exonuclease III enzymefrom E. coli.

SEQ ID NO: 10 shows the amino acid sequence of the exonuclease IIIenzyme from E. coli. This enzyme performs distributive digestion of 5′monophosphate nucleosides from one strand of double stranded DNA (dsDNA)in a 3′-5′ direction. Enzyme initiation on a strand requires a 5′overhang of approximately 4 nucleotides.

SEQ ID NO: 11 shows the codon optimised polynucleotide sequence derivedfrom the recJ gene from T. thermophilus. It encodes the RecJ enzyme fromT. thermophilus (TthRecJ-cd).

SEQ ID NO: 12 shows the amino acid sequence of the RecJ enzyme from T.thermophilus (TthRecJ-cd). This enzyme performs processive digestion of5′ monophosphate nucleosides from ssDNA in a 5′-3′ direction. Enzymeinitiation on a strand requires at least 4 nucleotides.

SEQ ID NO: 13 shows the codon optimised polynucleotide sequence derivedfrom the bacteriophage lambda exo (redX) gene. It encodes thebacteriophage lambda exonuclease.

SEQ ID NO: 14 shows the amino acid sequence of the bacteriophage lambdaexonuclease. The sequence is one of three identical subunits thatassemble into a trimer. The enzyme performs highly processive digestionof nucleotides from one strand of dsDNA, in a 5′-3′ direction(http://www.neb.com/nebecomm/products/productM0262.asp). Enzymeinitiation on a strand preferentially requires a 5′ overhang ofapproximately 4 nucleotides with a 5′ phosphate.

SEQ ID NOs: 15 to 17 show the amino acid sequences of the mature formsof the MspB, C and D mutants respectively. The mature forms lack thesignal sequence.

SEQ ID NOs: 18 to 32 show the sequences used in the Examples.

SEQ ID NO: 33 shows the polynucleotide sequence encoding one subunit ofα-HL-Q.

SEQ ID NO: 34 shows the amino acid sequence of one subunit of α-HL-Q.

SEQ ID NO: 35 shows the polynucleotide sequence encoding one subunit ofα-HL-E287C-QC-D5FLAGH6.

SEQ ID NO: 36 shows the amino acid sequence of one subunit ofα-HL-E287C-QC-D5FLAGH6.

SEQ ID NO: 37 shows the polynucleotide sequence encoding one subunit ofα-hemolysin-E111N/K147N (α-HL-NN; Stoddart et al., PNAS, 2009; 106(19):7702-7707).

SEQ ID NO: 38 shows the amino acid sequence of one subunit of α-HL-NN.

SEQ ID NO: 39 shows the sequence used in Example 5.

SEQ ID NO: 40 and 41 show the sequences used in Example 6.

DETAILED DESCRIPTION OF THE INVENTION

It is to be understood that different applications of the disclosedproducts and methods may be tailored to the specific needs in the art.It is also to be understood that the terminology used herein is for thepurpose of describing particular embodiments of the invention only, andis not intended to be limiting.

In addition as used in this specification and the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontent clearly dictates otherwise. Thus, for example, reference to “ananalyte” includes two or more analytes, reference to “a detector”includes two or more such detectors, reference to “a pore” includes twoor more such pores, reference to “a nucleic acid sequence” includes twoor more such sequences, and the like.

All publications, patents and patent applications cited herein, whethersupra or infra, are hereby incorporated by reference in their entirety.

Methods of the Invention

The invention provides a method for determining the presence, absence orcharacteristics of an analyte. The method comprises coupling the analyteto a membrane and allowing the analyte to interact with a detectorpresent in the membrane. The presence, absence or characteristics of theanalyte is thereby determined. In one embodiment, the invention providesa method for determining the presence or absence of an analyte,comprising (a) coupling the analyte to a membrane and (b) allowing theanalyte to interact with a detector present in the membrane and therebydetermining the presence or absence of the analyte.

As discussed above, coupling the analyte to a membrane containing thedetector lowers by several orders of magnitude the amount of analyterequired. The method is of course advantageous for detecting analytesthat are present at low concentrations. The method preferably allows thepresence or characteristics of the analyte to be determined when theanalyte is present at a concentration of from about 0.001 pM to about 1nM, such as less than 0.01 pM, less than 0.1 pM, less than 1 pM, lessthan 10 pM or less than 100 pM.

The method of the invention is particularly advantageous for nucleicacid sequencing because, as discussed above, only small amounts ofpurified nucleic acid can be obtained from human blood. The methodpreferably allows estimating the sequence of, or allows sequencing of, atarget polynucleotide that is present at a concentration of from about0.001 pM to about 1 nM, such as less than 0.01 pM, less than 0.1 pM,less than 1 pM, less than 10 pM or less than 100 pM.

Coupling one end of a polynucleotide to the membrane (even temporarily)also means that the end will be prevented from interfering with thenanopore-based sequencing process. This is discussed in more detailbelow with reference to the Exonuclease Sequencing method of theinvention.

The method of the invention may comprise determining or measuring one ormore characteristics of an analyte, such as a polynucleotide. The methodmay involve determining or measuring two, three, four or five or morecharacteristics of the analyte, such as a polynucleotide. Forpolynucleotides, the one or more characteristics are preferably selectedfrom (i) the length of the target polynucleotide, (ii) the identity ofthe target polynucleotide, (iii) the sequence of the targetpolynucleotide, (iv) the secondary structure of the targetpolynucleotide and (v) whether or not the target polynucleotide ismodified. Any combination of (i) to (v) may be determined or measured inaccordance with the invention. The method preferably comprisesestimating the sequence of or sequencing a polynucleotide.

Analyte

The analyte can be any substance. Suitable analytes include, but are notlimited to, metal ions, inorganic salts, polymers, such as a polymericacids or bases, dyes, bleaches, pharmaceuticals, diagnostic agents,recreational drugs, explosives and environmental pollutants.

The analyte can be an analyte that is secreted from cells.Alternatively, the analyte can be an analyte that is present insidecells such that the analyte must be extracted from the cells before theinvention can be carried out.

The analyte is preferably an amino acid, peptide, polypeptide, a proteinor a polynucleotide. The amino acid, peptide, polypeptide or protein canbe naturally-occurring or non-naturally-occurring. The polypeptide orprotein can include within it synthetic or modified amino acids. Anumber of different types of modification to amino acids are known inthe art. For the purposes of the invention, it is to be understood thatthe analyte can be modified by any method available in the art.

The protein can be an enzyme, antibody, hormone, growth factor or growthregulatory protein, such as a cytokine. The cytokine may be selectedfrom an interleukin, preferably IFN-1, IL-2, IL-4, IL-5, IL-6, IL-10,IL-12 or IL-13, an interferon, preferably IL-γ or other cytokines suchas TNF-α. The protein may be a bacterial protein, fungal protein, virusprotein or parasite-derived protein. Before it is contacted with thepore or channel, the protein may be unfolded to form a polypeptidechain.

The analyte is most preferably a polynucleotide, such as a nucleic acid.Polynucleotides are discussed in more detail below. A polynucleotide maybe coupled to the membrane at its 5′ end or 3′ end or at one or moreintermediate points along the strand. The polynucleotide can be singlestranded or double stranded as discussed below. The polynucleotide maybe circular. The polynucleotide may be an aptamer, a probe whichhybridises to microRNA or microRNA itself (Wang, Y. et al, NatureNanotechnology, 2011, 6, 668-674).

When the analyte is a probe which hybridises to microRNA, the probe maybe coupled permanently (FIG. 23A) or transiently (FIGS. 23 B and C) tothe membrane. The probe itself may be adapted to couple directly to themembrane or may hybridise to a complementary polynucleotide which hasbeen adapted to couple to the membrane. The analyte may be a complex ofmicroRNA hybridised to a probe where the probe has distinctive sequencesor barcodes enabling it to be identified unambiguously.

When the analyte is an aptamer, the aptamer may be coupled permanentlyor transiently to the membrane. The aptamer itself may be adapted tocouple directly to the membrane or may hybridise to a complementarypolynucleotide which has been adapted to couple to the membrane. Theaptamer may be bound or unbound to a protein analyte and the ultimatepurpose of detecting the aptamer may be to detect the presence, absenceor characteristics of a protein analyte to which it binds.

The analyte is present in any suitable sample. The invention istypically carried out on a sample that is known to contain or suspectedto contain the analyte. The invention may be carried out on a samplethat contains one or more analytes whose identity is unknown.Alternatively, the invention may be carried out on a sample to confirmthe identity of one or more analytes whose presence in the sample isknown or expected.

The sample may be a biological sample. The invention may be carried outin vitro on a sample obtained from or extracted from any organism ormicroorganism. The organism or microorganism is typically archaean,prokaryotic or eukaryotic and typically belongs to one the fivekingdoms: plantae, animalia, fungi, monera and protista. The inventionmay be carried out in vitro on a sample obtained from or extracted fromany virus. The sample is preferably a fluid sample. The sample typicallycomprises a body fluid of the patient. The sample may be urine, lymph,saliva, mucus or amniotic fluid but is preferably blood, plasma orserum. Typically, the sample is human in origin, but alternatively itmay be from another mammal animal such as from commercially farmedanimals such as horses, cattle, sheep or pigs or may alternatively bepets such as cats or dogs. Alternatively a sample of plant origin istypically obtained from a commercial crop, such as a cereal, legume,fruit or vegetable, for example wheat, barley, oats, canola, maize,soya, rice, bananas, apples, tomatoes, potatoes, grapes, tobacco, beans,lentils, sugar cane, cocoa, cotton.

The sample may be a non-biological sample. The non-biological sample ispreferably a fluid sample. Examples of a non-biological sample includesurgical fluids, water such as drinking water, sea water or river water,and reagents for laboratory tests.

The sample is typically processed prior to being assayed, for example bycentrifugation or by passage through a membrane that filters outunwanted molecules or cells, such as red blood cells. The sample may bemeasured immediately upon being taken. The sample may also be typicallystored prior to assay, preferably below −70° C.

Membrane

Any membrane may be used in accordance with the invention. Suitablemembranes are well-known in the art. The membrane is preferably anamphiphilic layer. An amphiphilic layer is a layer formed fromamphiphilic molecules, such as phospholipids, which have bothhydrophilic and lipophilic properties. The amphiphilic molecules may besynthetic or naturally occurring. Non-naturally occurring amphiphilesand amphiphiles which form a monolayer are known in the art and include,for example, block copolymers (Gonzalez-Perez et al., Langmuir, 2009,25, 10447-10450). Block copolymers are polymeric materials in which twoor more monomer sub-units that are polymerized together to create asingle polymer chain. Block copolymers typically have properties thatare contributed by each monomer sub-unit. However, a block copolymer mayhave unique properties that polymers formed from the individualsub-units do not possess. Block copolymers can be engineered such thatone of the monomer sub-units is hydrophobic (i.e. lipophilic), whilstthe other sub-unit(s) are hydrophilic whilst in aqueous media. In thiscase, the block copolymer may possess amphiphilic properties and mayform a structure that mimics a biological membrane. The block copolymermay be a diblock (consisting of two monomer sub-units), but may also beconstructed from more than two monomer sub-units to form more complexarrangements that behave as amphipiles. The copolymer may be a triblock,tetrablock or pentablock copolymer.

Archaebacterial bipolar tetraether lipids are naturally occurring lipidsthat are constructed such that the lipid forms a monolayer membrane.These lipids are generally found in extremophiles that survive in harshbiological environments, thermophiles, halophiles and acidophiles. Theirstability is believed to derive from the fused nature of the finalbilayer. It is straightforward to construct block copolymer materialsthat mimic these biological entities by creating a triblock polymer thathas the general motif hydrophilic-hydrophobic-hydrophilic. This materialmay form monomeric membranes that behave similarly to lipid bilayers andencompasse a range of phase behaviours from vesicles through to laminarmembranes. Membranes formed from these triblock copolymers hold severaladvantages over biological lipid membranes. Because the triblockcopolymer is synthesized, the exact construction can be carefullycontrolled to provide the correct chain lengths and properties requiredto form membranes and to interact with pores and other proteins.

Block copolymers may also be constructed from sub-units that are notclassed as lipid sub-materials; for example a hydrophobic polymer may bemade from siloxane or other non-hydrocarbon based monomers. Thehydrophilic sub-section of block copolymer can also possess low proteinbinding properties, which allows the creation of a membrane that ishighly resistant when exposed to raw biological samples. This head groupunit may also be derived from non-classical lipid head-groups.

Triblock copolymer membranes also have increased mechanical andenvironmental stability compared with biological lipid membranes, forexample a much higher operational temperature or pH range. The syntheticnature of the block copolymers provides a platform to customize polymerbased membranes for a wide range of applications.

In a preferred embodiment, the invention provides a method fordetermining the presence, absence or characteristics of an analyte,comprising (a) coupling the analyte to a membrane comprising a triblockcopolymer, optionally wherein the membrane is modified to facilitate thecoupling, and (b) allowing the analyte to interact with a detectorpresent in the membrane and thereby determining the presence, absence orcharacteristics of the analyte. As discussed above, a triblock copolymeris a polymer formed from three different monomer sub-units.

The amphiphilic molecules may be chemically-modified or functionalisedto facilitate coupling of the analyte.

The amphiphilic layer may be a monolayer or a bilayer. The amphiphiliclayer is typically planar. The amphiphilic layer may be curved.

Amphiphilic membranes are typically naturally mobile, essentially actingas two dimensional fluids with lipid diffusion rates of approximately10⁻⁸ cm s-1. This means that the detector and coupled analyte cantypically move within an amphiphilic membrane.

The membrane is preferably a lipid bilayer. Lipid bilayers are models ofcell membranes and serve as excellent platforms for a range ofexperimental studies. For example, lipid bilayers can be used for invitro investigation of membrane proteins by single-channel recording.Alternatively, lipid bilayers can be used as biosensors to detect thepresence of a range of substances. The lipid bilayer may be any lipidbilayer. Suitable lipid bilayers include, but are not limited to, aplanar lipid bilayer, a supported bilayer or a liposome. The lipidbilayer is preferably a planar lipid bilayer. Suitable lipid bilayersare disclosed in International Application No. PCT/GB08/000563(published as WO 2008/102121), International Application No.PCT/GB08/004127 (published as WO 2009/077734) and InternationalApplication No. PCT/GB2006/001057 (published as WO 2006/100484).

Methods for forming lipid bilayers are known in the art. Suitablemethods are disclosed in the Example. Lipid bilayers are commonly formedby the method of Montal and Mueller (Proc. Natl. Acad. Sci. USA., 1972;69: 3561-3566), in which a lipid monolayer is carried on aqueoussolution/air interface past either side of an aperture which isperpendicular to that interface. The lipid is normally added to thesurface of an aqueous electrolyte solution by first dissolving it in anorganic solvent and then allowing a drop of the solvent to evaporate onthe surface of the aqueous solution on either side of the aperture. Oncethe organic solvent has evaporated, the solution/air interfaces oneither side of the aperture are physically moved up and down past theaperture until a bilayer is formed. Planar lipid bilayers may be formedacross an aperture in a membrane or across an opening into a recess.

The method of Montal & Mueller is popular because it is a cost-effectiveand relatively straightforward method of forming good quality lipidbilayers that are suitable for protein pore insertion. Other commonmethods of bilayer formation include tip-dipping, painting bilayers andpatch-clamping of liposome bilayers.

Tip-dipping bilayer formation entails touching the aperture surface (forexample, a pipette tip) onto the surface of a test solution that iscarrying a monolayer of lipid. Again, the lipid monolayer is firstgenerated at the solution/air interface by allowing a drop of lipiddissolved in organic solvent to evaporate at the solution surface. Thebilayer is then formed by the Langmuir-Schaefer process and requiresmechanical automation to move the aperture relative to the solutionsurface.

For painted bilayers, a drop of lipid dissolved in organic solvent isapplied directly to the aperture, which is submerged in an aqueous testsolution. The lipid solution is spread thinly over the aperture using apaintbrush or an equivalent. Thinning of the solvent results information of a lipid bilayer. However, complete removal of the solventfrom the bilayer is difficult and consequently the bilayer formed bythis method is less stable and more prone to noise duringelectrochemical measurement.

Patch-clamping is commonly used in the study of biological cellmembranes. The cell membrane is clamped to the end of a pipette bysuction and a patch of the membrane becomes attached over the aperture.The method has been adapted for producing lipid bilayers by clampingliposomes which then burst to leave a lipid bilayer sealing over theaperture of the pipette. The method requires stable, giant andunilamellar liposomes and the fabrication of small apertures inmaterials having a glass surface.

Liposomes can be formed by sonication, extrusion or the Mozafari method(Colas et al. (2007) Micron 38:841-847).

In a preferred embodiment, the lipid bilayer is formed as described inInternational Application No. PCT/GB08/004127 (published as WO2009/077734). Advantageously in this method, the lipid bilayer is formedfrom dried lipids. In a most preferred embodiment, the lipid bilayer isformed across an opening as described in WO2009/077734(PCT/GB08/004127).

A lipid bilayer is formed from two opposing layers of lipids. The twolayers of lipids are arranged such that their hydrophobic tail groupsface towards each other to form a hydrophobic interior. The hydrophilichead groups of the lipids face outwards towards the aqueous environmenton each side of the bilayer. The bilayer may be present in a number oflipid phases including, but not limited to, the liquid disordered phase(fluid lamellar), liquid ordered phase, solid ordered phase (lamellargel phase, interdigitated gel phase) and planar bilayer crystals(lamellar sub-gel phase, lamellar crystalline phase).

Any lipid composition that forms a lipid bilayer may be used. The lipidcomposition is chosen such that a lipid bilayer having the requiredproperties, such surface charge, ability to support membrane proteins,packing density or mechanical properties, is formed. The lipidcomposition can comprise one or more different lipids. For instance, thelipid composition can contain up to 100 lipids. The lipid compositionpreferably contains 1 to 10 lipids. The lipid composition may comprisenaturally-occurring lipids and/or artificial lipids.

The lipids typically comprise a head group, an interfacial moiety andtwo hydrophobic tail groups which may be the same or different. Suitablehead groups include, but are not limited to, neutral head groups, suchas diacylglycerides (DG) and ceramides (CM); zwitterionic head groups,such as phosphatidylcholine (PC), phosphatidylethanolamine (PE) andsphingomyelin (SM); negatively charged head groups, such asphosphatidylglycerol (PG); phosphatidylserine (PS), phosphatidylinositol(PI), phosphatic acid (PA) and cardiolipin (CA); and positively chargedheadgroups, such as trimethylammonium-Propane (TAP). Suitableinterfacial moieties include, but are not limited to,naturally-occurring interfacial moieties, such as glycerol-based orceramide-based moieties. Suitable hydrophobic tail groups include, butare not limited to, saturated hydrocarbon chains, such as lauric acid(n-Dodecanolic acid), myristic acid (n-Tetradecononic acid), palmiticacid (n-Hexadecanoic acid), stearic acid (n-Octadecanoic) and arachidic(n-Eicosanoic); unsaturated hydrocarbon chains, such as oleic acid(cis-9-Octadecanoic); and branched hydrocarbon chains, such asphytanoyl. The length of the chain and the position and number of thedouble bonds in the unsaturated hydrocarbon chains can vary. The lengthof the chains and the position and number of the branches, such asmethyl groups, in the branched hydrocarbon chains can vary. Thehydrophobic tail groups can be linked to the interfacial moiety as anether or an ester.

The lipids can also be chemically-modified. The head group or the tailgroup of the lipids may be chemically-modified. Suitable lipids whosehead groups have been chemically-modified include, but are not limitedto, PEG-modified lipids, such as1,2-Diacyl-sn-Glycero-3-Phosphoethanolamine-N- [Methoxy(Polyethyleneglycol)-2000]; functionalised PEG Lipids, such as1,2-Distearoyl-sn-Glycero-3 Phosphoethanolamine-N-[Biotinyl(PolyethyleneGlycol)2000]; and lipids modified for conjugation, such as1,2-Dioleoyl-sn-Glycero-3-Phosphoethanolamine-N-(succinyl) and1,2-Dipalmitoyl-sn-Glycero-3-Phosphoethanolamine-N-(Biotinyl). Suitablelipids whose tail groups have been chemically-modified include, but arenot limited to, polymerisable lipids, such as1,2-bis(10,12-tricosadiynoyl)-sn-Glycero-3-Phosphocholine; fluorinatedlipids, such as1-Palmitoyl-2-(16-Fluoropalmitoyl)-sn-Glycero-3-Phosphocholine;deuterated lipids, such as1,2-Dipalmitoyl-D62-sn-Glycero-3-Phosphocholine; and ether linkedlipids, such as 1,2-Di-O-phytanyl-sn-Glycero-3-Phosphocholine. Thelipids may be chemically-modified or functionalised to facilitatecoupling of the analyte.

The amphiphilic layer, for example the lipid composition, typicallycomprises one or more additives that will affect the properties of thelayer. Suitable additives include, but are not limited to, fatty acids,such as palmitic acid, myristic acid and oleic acid; fatty alcohols,such as palmitic alcohol, myristic alcohol and oleic alcohol; sterols,such as cholesterol, ergosterol, lanosterol, sitosterol andstigmasterol; lysophospholipids, such as1-Acyl-2-Hydroxy-sn-Glycero-3-Phosphocholine; and ceramides.

In another preferred embodiment, the membrane is a solid state layer. Asolid-state layer is not of biological origin. In other words, a solidstate layer is not derived from or isolated from a biologicalenvironment such as an organism or cell, or a synthetically manufacturedversion of a biologically available structure. Solid state layers can beformed from both organic and inorganic materials including, but notlimited to, microelectronic materials, insulating materials such asSi3N4, Al203, and SiO, organic and inorganic polymers such as polyamide,plastics such as Teflon® or elastomers such as two-componentaddition-cure silicone rubber, and glasses. The solid state layer may beformed from graphene. Suitable graphene layers are disclosed inInternational Application No. PCT/US2008/010637 (published as WO2009/035647).

Coupling

The analyte may be coupled to the membrane using any known method. Ifthe membrane is an amphiphilic layer, such as a lipid bilayer, theanalyte is preferably coupled to the membrane via a polypeptide presentin the membrane or a hydrophobic anchor present in the membrane. Thehydrophobic anchor is preferably a lipid, fatty acid, sterol, carbonnanotube, polypeptide, protein or amino acid, for example cholesterol,palmitate or tocopherol. In preferred embodiments, the analyte is notcoupled to the membrane via the detector.

The components of the membrane, such as the amphiphilic molecules orlipids, may be chemically-modified or functionalised to facilitatecoupling of the analyte to the membrane either directly or via one ormore linkers. Examples of suitable chemical modifications and suitableways of functionalising the components of the membrane are discussed inmore detail below. Any proportion of the membrane components may befunctionalized, for example at least 0.01%, at least 0.1%, at least 1%,at least 10%, at least 25%, at least 50% or 100%.

The analyte may be coupled directly to the membrane. The analyte may becoupled directly to the membrane at one or more, such as 2, 3, 4 ormore, points.

The analyte is preferably coupled to the membrane via a linker. Theanalyte may be coupled to the membrane via one or more, such as 2, 3, 4or more, linkers. One linker may couple more than one, such as 2, 3, 4or more, analytes to the membrane.

The analyte may be coupled to the membrane directly at one or morepoints and via one or more linkers.

Preferred linkers include, but are not limited to, polymers, such aspolynucleotides, polyethylene glycols (PEGs), polysaccharides andpolypeptides. These linkers may be linear, branched or circular. Forinstance, the linker may be a circular polynucleotide. If the analyte isitself a polynucleotide, it may hybridize to a complementary sequence onthe circular polynucleotide linker.

Functionalised linkers and the ways in which they can couple moleculesare known in the art. For instance, linkers functionalised withmaleimide groups will react with and attach to cysteine residues inproteins. In the context of this invention, the protein may be presentin the membrane, may be the analyte itself or may be used to bind to theanalyte. This is discussed in more detail below.

Crosslinkage of analytes can be avoided using a “lock and key”arrangement. Only one end of each linker may react together to form alonger linker and the other ends of the linker each react with theanalyte or membrane respectively. Such linkers are described inInternational Application No. PCT/GB10/000132 (published as WO2010/086602).

The use of a linker is preferred in the sequencing embodiments discussedbelow. If a polynucleotide analyte is permanently coupled directly tothe membrane, then some sequence data will be lost as the sequencing runcannot continue to the end of the polynucleotide due to the distancebetween the membrane and the detector. If a linker is used, then thepolynucleotide analyte can be processed to completion. The coupling maybe permanent or stable. In other words, the coupling may be such thatthe analyte remains coupled to the membrane during the method. Thecoupling may be transient. In other words, the coupling may be such thatthe analyte decouples from the membrane during the method. For certainapplications, such as aptamer detection, the transient nature of thecoupling is preferred. If a permanent or stable linker is attacheddirectly to either the 5′ or 3′ end of a polynucleotide and the linkeris shorter than the distance between the bilayer and the nanopore'schannel or the polynucleotide binding protein's active site, then somesequence data will be lost as the sequencing run cannot continue to theend of the polynucleotide. If the coupling is transient, then when thecoupled end randomly becomes free of the bilayer, then thepolynucleotide can be processed to completion. Chemical groups that formpermanent/stable or transient links with the membrane are discussed inmore detail below. The analyte may be transiently coupled to anamphiphilic layer or lipid bilayer using cholesterol or a fatty acylchain. Any fatty acyl chain having a length of from 6 to 30 carbon atom,such as hexadecanoic acid, may be used.

In preferred embodiments, a polynucleotide analyte, such as a nucleicacid, is coupled to an amphiphilic layer such as a lipid bilayer.Coupling of nucleic acids to synthetic lipid bilayers has been carriedout previously with various different tethering strategies. These aresummarised in Table 3 below.

TABLE 3 Attachment Type of group coupling Reference Thiol StableYoshina-Ishii, C. and S. G. Boxer (2003). “Arrays of mobile tetheredvesicles on supported lipid bilayers.” J Am Chem Soc 125(13): 3696-7.Biotin Stable Nikolov, V., R. Lipowsky, et al. (2007). “Behavior ofgiant vesicles with anchored DNA molecules.” Biophys J 92(12): 4356-68Cholestrol Transient Pfeiffer, I. and F. Hook (2004). “Bivalentcholesterol-based coupling of oligonucletides to lipid membraneassemblies.” J Am Chem Soc 126(33): 10224-5 Surfactant Stable vanLengerich, B., R. J. Rawle, et al. (eg. Lipid, “Covalent attachment oflipid vesicles to a Palmitate, fluid-supported bilayer allowsobservation of etc) DNA-mediated vesicle interactions.” Langmuir 26(11):8666-72

Synthetic polynucleotide analytes or linkers may be functionalised usinga modified phosphoramidite in the synthesis reaction, which is easilycompatible for the direct addition of suitable coupling moieties, suchas cholesterol, tocopherol or palmitate, as well as for reactive groups,such as thiol, cholesterol, lipid and biotin groups. These differentattachment chemistries give a suite of options for attachment to targetpolynucleotides. Each different modification group tethers thepolynucleotide in a slightly different way and coupling is not alwayspermanent so giving different dwell times for the analyte to thebilayer. The advantages of transient coupling are discussed above.

Coupling of polynucleotides to a linker or to a functionalised membranecan also be achieved by a number of other means provided that acomplementary reactive group or a tether can be added to the targetpolynucleotide. The addition of reactive groups to either end of DNA hasbeen reported previously. A thiol group can be added to the 5′ of ssDNAor dsDNA using T4 polynucleotide kinase and ATPγS (Grant, G. P. and P.Z. Qin (2007). “A facile method for attaching nitroxide spin labels atthe 5′ terminus of nucleic acids.” Nucleic Acids Res 35(10): e77). Anazide group could be added to the 5′-phosphate of ssDNA or dsDNA usingT4 polynucleotide kinase and γ-[2-Azidoethyl]-ATP orγ-[6-Azidohexyl]-ATP. Using thiol or Click chemistry a tether,containing either a thiol, iodoacetamide OPSS or maleimide group(reactive to thiols) or a DIBO (dibenzocyclooxtyne) or alkyne group(reactive to azides), can be covalently attached to the analyte. A morediverse selection of chemical groups, such as biotin, thiols andfluorophores, can be added using terminal transferase to incorporatemodified oligonucleotides to the 3′ of ssDNA (Kumar, A., P. Tchen, etal. (1988). “Nonradioactive labeling of synthetic oligonucleotide probeswith terminal deoxynucleotidyl transferase.” Anal Biochem 169(2):376-82). Example 3 below describes how DNA can be coupled to a lipidbilayer using streptavidin/biotin. Streptavidin/biotin coupling may beused for any other analyte. It may also be possible that tethers couldbe directly added to target polynucleotides using terminal transferasewith suitably modified nucleotides (eg. cholesterol or palmitate).

Alternatively, the reactive group or tether could be considered to bethe addition of a short piece of polynucleotide, such as DNA,complementary to one already coupled to the bilayer, so that attachmentcan be achieved via hybridisation. In this case, the reactive group maybe a single strand or double strand polynucleotide. The reactive groupmay be ligated to a single strand or double strand polynucleotideanalyte. Ligation of short pieces of ssDNA have been reported using T4RNA ligase I (Troutt, A. B., M. G. McHeyzer-Williams, et al. (1992).“Ligation-anchored PCR: a simple amplification technique withsingle-sided specificity.” Proc Natl Acad Sci USA 89(20): 9823-5).Alternatively, either ssDNA or dsDNA could be ligated to native analytedsDNA and then the two strands separated by thermal or chemicaldenaturation. To native dsDNA, it is possible to add either a piece ofssDNA to one or both of the ends of the duplex, or dsDNA to one or bothends. For addition of single stranded nucleic acids to the native DNAthis can be achieved using T4 RNA ligase I as for ligation to otherregions of single stranded nucleic acids. For addition of dsDNA tonative duplex DNA then ligation can be “blunt-ended”, with complementary3′ dA/dT tails on the native DNA and adapter respectively (as isroutinely done for many sample prep applications to prevent concatemeror dimer formation) or using “sticky-ends” generated by restrictiondigestion of the native DNA and ligation of compatible adapters. Then,when the duplex is melted, each single strand will have either a 5′ or3′ modification if ssDNA was used for ligation or a modification at the5′ end, the 3′ end or both if dsDNA was used for ligation. If thepolynucleotide is a synthetic strand, the coupling chemistry can beincorporated during the chemical synthesis of the polynucleotide. Forinstance, the polynucleotide can be synthesised using a primer having areactive group attached to it.

Adenylated nucleic acids (AppDNA) are intermediates in ligationreactions, where an adenosine-monophostate is attached to the5′-phosphate of the nucleic acid. Various kits are available forgeneration of this intermediate, such as the 5′ DNA Adenylation Kit fromNEB. By substituting ATP in the reaction for a modifided nucleotidetriphosphate, then addition of reactive groups (such as thiols, amines,biotin, azides, etc) to the 5′ of DNA should be possible. It may also bepossible that tethers could be directly added to target polynucleotidesusing a 5′ DNA adenylation kit with suitably modified nucleotides (e.g.cholesterol or palmitate).

A common technique for the amplification of sections of genomic DNA isusing polymerase chain reaction (PCR). Here, using two syntheticoligonucleotide primers, a number of copies of the same section of DNAcan be generated, where for each copy the 5′ of each strand in theduplex will be a synthetic polynucleotide. By using an antisense primersingle or multiple nucleotides can be added to 3′ end of single ordouble stranded DNA by employing a polymerase. Examples of polymeraseswhich could be used include, but are not limited to, TerminalTransferase, Klenow and E. coli Poly(A) polymerase). By substituting ATPin the reaction for a modified nucleotide triphosphate then reactivegroups, such as a cholesterol, thiol, amine, azide, biotin or lipid, canbe incorporated into the DNA. Therefore, each copy of the targetamplified DNA will contain a reactive group for coupling.

Ideally, the analyte is coupled to the membrane without having tofunctionalise the analyte. This can be achieved by anchoring a bindinggroup, such as a polynucleotide binding protein or a chemical group, tothe membrane and allowing the binding group to interact with the analyteor by functionalizing the membrane. The binding group may be coupled tothe membrane by any of the methods described herein. In particular, thebinding group may be coupled to the membrane using one or more linkers,such as maleimide functionalised linkers.

In this embodiment, the analyte is typically RNA, DNA, PNA, TNA or LNAand may be double or single stranded. This embodiment is particularlysuited to genomic DNA analytes.

The binding group can be any group that interacts with single or doublestranded nucleic acids, specific nucleotide sequences within the analyteor patterns of modified nucleotides within the analyte, or any otherligand that is present on the polynucleotide.

Suitable binding proteins include E. coli single stranded bindingprotein, P5 single stranded binding protein, T4 gp32 single strandedbinding protein, the TOPO V dsDNA binding region, human histoneproteins, E. coli HU DNA binding protein and other archaeal, prokaryoticor eukaryotic single- or double-stranded nucleic acid binding proteins,including those listed below.

The specific nucleotide sequences could be sequences recognised bytranscription factors, ribosomes, endonucleases, topoisomerases orreplication initiation factors. The patterns of modified nucleotidescould be patterns of methylation or damage.

The chemical group can be any group which intercalates with or interactswith a polynucleotide analyte. The group may intercalate or interactwith the polynucleotide analyte via electrostatic, hydrogen bonding orVan der Waals interactions. Such groups include a lysine monomer,poly-lysine (which will interact with ssDNA or dsDNA), ethidium bromide(which will intercalate with dsDNA), universal bases or universalnucleotides (which can hybridise with any polynucleotide analyte) andosmium complexes (which can react to methylated bases). A polynucleotideanalyte may therefore be coupled to the membrane using one or moreuniversal nucleotides attached to the membrane. Each universalnucleotide residue may be attached to the membrane using one or morelinkers. Examples of universal bases include inosine, 3-nitropyrrole,5-nitroindole, 4-nitroindole, 6-nitroindole,3,4-dihydro-pyrimido[4,5-c][1,2]oxazin-7-one (dP),2-dimethylaminomethyleneamino-6-methyoxyaminopurine (dK), deoxy inosine,deoxy nebularine.

In this embodiment at least 1%, at least 10%, at least 25%, at least 50%or 100% of the membrane components may be functionalized.

Where the binding group is a protein, it may be able to anchor directlyinto the membrane without further functonalisation, for example if italready has an external hydrophobic region which is compatible with themembrane. Examples of such proteins include transmembrane proteins.Alternatively the protein may be expressed with a genetically fusedhydrophobic region which is compatible with the membrane. Suchhydrophobic protein regions are know in the art

The binding group is preferably mixed with the analyte before contactingwith the membrane, but the binding group may be contacted with themembrane and subsequently contacted with the analyte.

In another aspect the analyte may be functionalised, using methodsdescribed above, so that it can be recognised by a specific bindinggroup. Specifically the analyte may be functionalised with a ligand suchas biotin (for binding to streptavidin), amylose (for binding to maltosebinding protein or a fusion protein), Ni-NTA (for binding topoly-histidine or poly-histidine tagged proteins) or a peptides (such asan antigen),

According to a further aspect, the binding group may be used to couplepolynucleotide analyte to the membrane when the analyte has bound to apolynucleotide adapter. Specifically the analyte binds to an adaptorwhich comprises a leader sequence designed to preferentially thread intoa detector such as a nanopore. Such a leader sequence may comprise ahomopolymeric polynucleotide or an abasic region. The adaptor typicallyis designed to hybridise to a linker and to ligate to or hybridise tothe analyte. This creates competition between the analyte and theadaptor to enter the detector. If the linker comprises a binding group,the greater length of the analyte compared to the adapter means thatseveral linkers can bind to the analyte simultaneously, thus increasingthe concentration of analyte relative to that of the adapter.

Any of the methods discussed above for coupling polynucleotides toamphiphilic layers, such as lipid bilayers, can of course be applied toother analyte and membrane combinations. In some embodiments, an aminoacid, peptide, polypeptide or protein is coupled to a lipid bilayer.Various methodologies for the chemical attachment of such analytes areavailable. An example of a molecule used in chemical attachment is EDC(1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride). Reactivegroups can also be added to the 5′ of DNA using commercially availablekits (Thermo Pierce, Part No. 22980). Suitable methods include, but arenot limited to, transient affinity attachment using histidine residuesand Ni-NTA, as well as more robust covalent attachment by reactivecysteines, lysines or non natural amino acids.

Detector

The detector can be any structure that provides a readable signal inresponse to the presence, the absence or the characteristics of theanalyte. The detector can be any structure that provides a readablesignal in response to the presence or the absence of the analyte.Suitable detectors are known in the art. They include, but are notlimited to transmembrane pores, tunnelling electrodes, classiselectrodes, nanotubes, FETs (field-effect transistors) and opticaldetectors, such as atomic force microscopes (AFMs) and scanningtunneling microscopes (STMs).

In preferred embodiments, the detector detects the analyte usingelectrical means. Electrical measurements may be made using standardsingle channel recording equipment as describe in Stoddart D et al.,Proc Natl Acad Sci, 12; 106(19):7702-7, Lieberman K R et al, J Am Chem.Soc. 2010; 132(50):17961-72, and International ApplicationWO-2000/28312. Alternatively, electrical measurements may be made usinga multi-channel system, for example as described in InternationalApplication WO-2009/077734 and International Application WO-2011/067559.

In other preferred embodiments, the detector does not detect the analyteusing fluorescent means.

The detector preferably comprises a transmembrane pore. A transmembranepore is a structure that permits hydrated ions driven by an appliedpotential to flow from one side of the membrane to the other side of themembrane.

The transmembrane pore is preferably a transmembrane protein pore. Atransmembrane protein pore is a polypeptide or a collection ofpolypeptides that permits hydrated ions, such as analyte, to flow fromone side of a membrane to the other side of the membrane. In the presentinvention, the transmembrane protein pore is capable of forming a porethat permits hydrated ions driven by an applied potential to flow fromone side of the membrane to the other. The transmembrane protein porepreferably permits analyte such as nucleotides to flow from one side ofthe membrane, such as a lipid bilayer, to the other. The transmembraneprotein pore preferably allows a polynucleotide or nucleic acid, such asDNA or RNA, to be move through the pore.

The transmembrane protein pore may be a monomer or an oligomer. The poreis preferably made up of several repeating subunits, such as 6, 7 or 8subunits. The pore is more preferably a heptameric or octameric pore.

The transmembrane protein pore typically comprises a barrel or channelthrough which the ions may flow. The subunits of the pore typicallysurround a central axis and contribute strands to a transmembrane βbarrel or channel or a transmembrane α-helix bundle or channel.

The barrel or channel of the transmembrane protein pore typicallycomprises amino acids that facilitate interaction with analyte, such asnucleotides, polynucleotides or nucleic acids. These amino acids arepreferably located near a constriction of the barrel or channel. Thetransmembrane protein pore typically comprises one or more positivelycharged amino acids, such as arginine, lysine or histidine, or aromaticamino acids, such as tyrosine or tryptophan. These amino acids typicallyfacilitate the interaction between the pore and nucleotides,polynucleotides or nucleic acids. The nucleotide detection can befacilitated with an adaptor. This is discussed in more detail below.

Transmembrane protein pores for use in accordance with the invention canbe derived from β-barrel pores or α-helix bundle pores. β-barrel porescomprise a barrel or channel that is formed from β-strands. Suitableβ-barrel pores include, but are not limited to, β-toxins, such asα-hemolysin, anthrax toxin and leukocidins, and outer membraneproteins/porins of bacteria, such as Mycobacterium smegmatis porin(Msp), for example MspA, MspB, MspC or MspD, outer membrane porin F(OmpF), outer membrane porin G (OmpG), outer membrane phospholipase Aand Neisseria autotransporter lipoprotein (NalP). α-helix bundle porescomprise a barrel or channel that is formed from α-helices. Suitableα-helix bundle pores include, but are not limited to, inner membraneproteins and a outer membrane proteins, such as WZA and ClyA toxin. Thetransmembrane pore may be derived from Msp or from α-hemolysin (α-HL).

For Strand Sequencing, the transmembrane protein pore is preferablyderived from Msp, preferably from MspA. Such a pore will be oligomericand typically comprises 7, 8, 9 or 10 monomers derived from Msp. Thepore may be a homo-oligomeric pore derived from Msp comprising identicalmonomers. Alternatively, the pore may be a hetero-oligomeric porederived from Msp comprising at least one monomer that differs from theothers. The pore may also comprise one or more constructs which comprisetwo or more covalently attached monomers derived from Msp. Suitablepores are disclosed in International Application No. PCT/GB2012/050301(claiming priority from U.S. Provisional Application No. 61/441,718).Preferably the pore is derived from MspA or a homolog or paralogthereof.

A monomer derived from Msp comprises the sequence shown in SEQ ID NO: 2or a variant thereof. SEQ ID NO: 2 is the NNN-RRK mutant of the MspAmonomer. It includes the following mutations: D90N, D91N, D93N, D118R,D134R and E139K. A variant of SEQ ID NO: 2 is a polypeptide that has anamino acid sequence which varies from that of SEQ ID NO: 2 and whichretains its ability to form a pore. The ability of a variant to form apore can be assayed using any method known in the art. For instance, thevariant may be inserted into a lipid bilayer along with otherappropriate subunits and its ability to oligomerise to form a pore maybe determined. Methods are known in the art for inserting subunits intomembranes, such as lipid bilayers. For example, subunits may besuspended in a purified form in a solution containing a lipid bilayersuch that it diffuses to the lipid bilayer and is inserted by binding tothe lipid bilayer and assembling into a functional state. Alternatively,subunits may be directly inserted into the membrane using the “pick andplace” method described in M. A. Holden, H. Bayley. J. Am. Chem. Soc.2005, 127, 6502-6503 and International Application No. PCT/GB2006/001057(published as WO 2006/100484).

Preferred variants are disclosed in International Application No.PCT/GB2012/050301 (claiming priority from U.S. Provisional ApplicationNo. 61/441,718). Particularly preferred variants include, but are notlimited to, those comprising the following substitution(s): L88N; L88S;L88Q; L88T; D90S; D90Q; D90Y; I105L; I105S; Q126R; G75S; G77S; G75S,G77S, L88N and Q126R; G75S, G77S, L88N, D90Q and Q126R; D90Q and Q126R;L88N, D90Q and Q126R; L88S and D90Q; L88N and D90Q; E59R; G75Q; G75N;G75S; G75T; G77Q; G77N; G77S; G77T; I78L; S81N; T83N; N86S; N86T; I87F;I87V; I87L; L88N; L88S; L88Y; L88F; L88V; L88Q; L88T; I89F; I89V; I89L;N90S; N90Q; N90L; N90Y; N91S; N91Q; N91L; N91M; N91I; N91A; N91V; N91G;G92A; G92S; N93S; N93A; N93T; I94L; T95V; A96R; A96D; A96V; A96N; A96S;A96T; P97S; P98S; F99S; G100S; L101F; N102K; N102S; N102T; S103A; S103Q;S103N; S103G; S103T; V104I; I105Y; I105L; I105A; I105Q; I105N; I105S;I105T; T106F; T1061; T106V; T106S; N108P; N108S; D90Q and I105A; D90Sand G92S; L88T and D90S; 187Q and D90S; 189Y and D90S; L88N and 189F;L88N and 189Y; D90S and G92A; D90S and I94N; D90S and V1041; L88D andI105K; L88N and Q126R; L88N, D90Q and D91R; L88N, D90Q and D91S; L88N,D90Q and I105V; D90Q, D93S and I105A; N91Y; N90Y and N91G; N90G andN91Y; N90G and N91G; I05G; N90R; N91R; N90R and N91R; N90K; N91K; N90Kand N91K; N90Q and N91G; N90G and N91Q; N90Q and N91Q; R118N; N91C;N90C; N90W; N91W; N90K; N91K; N90R; N91R; N90S and N91S; N90Y and I105A;N90G and I105A; N90Q and I105A; N90S and I105A; L88A and I105A; L88S andI105S; L88N and I105N; N90G and N93G; N90G; N93G; N90G and N91A; I105K;I105R; I105V; I105P; I105W; L88R; L88A; L88G; L88N; N90R and I105A; N90Sand I105A; L88A and I105A; L88S and I105S; L88N and I105N; L88C; S103C;and I105C.

In addition to the specific mutations discussed above, the variant mayinclude other mutations. Over the entire length of the amino acidsequence of SEQ ID NO: 2, a variant will preferably be at least 50%homologous to that sequence based on amino acid identity. Morepreferably, the variant may be at least 55%, at least 60%, at least 65%,at least 70%, at least 75%, at least 80%, at least 85%, at least 90% andmore preferably at least 95%, 97% or 99% homologous based on amino acididentity to the amino acid sequence of SEQ ID NO: 2 over the entiresequence. There may be at least 80%, for example at least 85%, 90% or95%, amino acid identity over a stretch of 100 or more, for example 125,150, 175 or 200 or more, contiguous amino acids (“hard homology”).

Standard methods in the art may be used to determine homology. Forexample the UWGCG Package provides the BESTFIT program which can be usedto calculate homology, for example used on its default settings(Devereux et al (1984) Nucleic Acids Research 12, p387-395). The PILEUPand BLAST algorithms can be used to calculate homology or line upsequences (such as identifying equivalent residues or correspondingsequences (typically on their default settings)), for example asdescribed in Altschul S. F. (1993) J Mol Evol 36:290-300; Altschul, S. Fet al (1990) J Mol Biol 215:403-10. Software for performing BLASTanalyses is publicly available through the National Center forBiotechnology Information (http://www.ncbi.nlm.nih.gov/).

SEQ ID NO: 2 is the NNN-RRK mutant of the MspA monomer. The variant maycomprise any of the mutations in the MspB, C or D monomers compared withMspA. The mature forms of MspB, C and D are shown in SEQ ID NOs: 15 to17. In particular, the variant may comprise the following substitutionpresent in MspB: A138P. The variant may comprise one or more of thefollowing substitutions present in MspC: A96G, N102E and A138P. Thevariant may comprise one or more of the following mutations present inMspD: Deletion of G1, L2V, E5Q, L8V, D13G, W21A, D22E, K47T, I49H, I68V,D91G, A96Q, N102D, S103T, V104I, S136K and G141A. The variant maycomprise combinations of one or more of the mutations and substitutionsfrom Msp B, C and D.

Amino acid substitutions may be made to the amino acid sequence of SEQID NO: 2 in addition to those discussed above, for example up to 1, 2,3, 4, 5, 10, 20 or 30 substitutions. Conservative substitutions replaceamino acids with other amino acids of similar chemical structure,similar chemical properties or similar side-chain volume. The aminoacids introduced may have similar polarity, hydrophilicity,hydrophobicity, basicity, acidity, neutrality or charge to the aminoacids they replace. Alternatively, the conservative substitution mayintroduce another amino acid that is aromatic or aliphatic in the placeof a pre-existing aromatic or aliphatic amino acid. Conservative aminoacid changes are well-known in the art and may be selected in accordancewith the properties of the 20 main amino acids as defined in Table 4below. Where amino acids have similar polarity, this can also bedetermined by reference to the hydropathy scale for amino acid sidechains in Table 5.

TABLE 4 Chemical properties of amino acids Ala aliphatic, hydrophobic,Met hydrophobic, neutral neutral Cys polar, hydrophobic, Asn polar,hydrophilic, neutral neutral Asp polar, hydrophilic, Pro hydrophobic,charged (−) neutral Glu polar, hydrophilic, Gln polar, hydrophilic,charged (−) neutral Phe aromatic, hydrophobic, Arg polar, hydrophilic,neutral charged (+) Gly aliphatic, neutral Ser polar, hydrophilic,neutral His aromatic, polar, Thr polar, hydrophilic, hydrophilic,charged (+) neutral Ile aliphatic, hydrophobic, Val aliphatic,hydrophobic, neutral neutral Lys polar, hydrophilic, Trp aromatic,hydrophobic, charged(+) neutral Leu aliphatic, hydrophobic, Tyraromatic, polar, neutral hydrophobic

TABLE 5 Hydropathy scale Side Chain Hydropathy Ile 4.5 Val 4.2 Leu 3.8Phe 2.8 Cys 2.5 Met 1.9 Ala 1.8 Gly −0.4 Thr −0.7 Ser −0.8 Trp −0.9 Tyr−1.3 Pro −1.6 His −3.2 Glu −3.5 Gln −3.5 Asp −3.5 Asn −3.5 Lys −3.9 Arg−4.5

One or more amino acid residues of the amino acid sequence of SEQ ID NO:2 may additionally be deleted from the polypeptides described above. Upto 1, 2, 3, 4, 5, 10, 20 or 30 residues may be deleted, or more.

Variants may include fragments of SEQ ID NO: 2. Such fragments retainpore forming activity. Fragments may be at least 50, 100, 150 or 200amino acids in length. Such fragments may be used to produce the pores.A fragment preferably comprises the pore forming domain of SEQ ID NO: 2.Fragments must include one of residues 88, 90, 91, 105, 118 and 134 ofSEQ ID NO: 2. Typically, fragments include all of residues 88, 90, 91,105, 118 and 134 of SEQ ID NO: 2.

One or more amino acids may be alternatively or additionally added tothe polypeptides described above. An extension may be provided at theamino terminal or carboxy terminal of the amino acid sequence of SEQ IDNO: 2 or polypeptide variant or fragment thereof. The extension may bequite short, for example from 1 to 10 amino acids in length.Alternatively, the extension may be longer, for example up to 50 or 100amino acids. A carrier protein may be fused to an amino acid sequenceaccording to the invention. Other fusion proteins are discussed in moredetail below.

As discussed above, a variant is a polypeptide that has an amino acidsequence which varies from that of SEQ ID NO: 2 and which retains itsability to form a pore. A variant typically contains the regions of SEQID NO: 2 that are responsible for pore formation. The pore formingability of Msp, which contains a β-barrel, is provided by β-sheets ineach subunit. A variant of SEQ ID NO: 2 typically comprises the regionsin SEQ ID NO: 2 that form β-sheets. One or more modifications can bemade to the regions of SEQ ID NO: 2 that form β-sheets as long as theresulting variant retains its ability to form a pore. A variant of SEQID NO: 2 preferably includes one or more modifications, such assubstitutions, additions or deletions, within its α-helices and/or loopregions.

The monomers derived from Msp may be modified to assist theiridentification or purification, for example by the addition of histidineresidues (a hist tag), aspartic acid residues (an asp tag), astreptavidin tag or a flag tag, or by the addition of a signal sequenceto promote their secretion from a cell where the polypeptide does notnaturally contain such a sequence. An alternative to introducing agenetic tag is to chemically react a tag onto a native or engineeredposition on the pore. An example of this would be to react a gel-shiftreagent to a cysteine engineered on the outside of the pore. This hasbeen demonstrated as a method for separating hemolysin hetero-oligomers(Chem. Biol. 1997 July; 4(7):497-505).

The monomer derived from Msp may be labelled with a revealing label. Therevealing label may be any suitable label which allows the pore to bedetected. Suitable labels include, but are not limited to fluorescentmolecules, radioisotopes, e.g. ¹²⁵I, ³⁵S, enzymes, antibodies, antigens,polynucleotides and ligands such as biotin.

The monomer derived from Msp may also be produced using D-amino acids.For instance, the monomer derived from Msp may comprise a mixture ofL-amino acids and D-amino acids. This is conventional in the art forproducing such proteins or peptides.

The monomer derived from Msp contains one or more specific modificationsto facilitate nucleotide discrimination. The monomer derived from Mspmay also contain other non-specific modifications as long as they do notinterfere with pore formation. A number of non-specific side chainmodifications are known in the art and may be made to the side chains ofthe monomer derived from Msp. Such modifications include, for example,reductive alkylation of amino acids by reaction with an aldehydefollowed by reduction with NaBH₄, amidination with methylacetimidate oracylation with acetic anhydride.

The monomer derived from Msp can be produced using standard methodsknown in the art. The monomer derived from Msp may be made syntheticallyor by recombinant means. For example, the pore may be synthesised by invitro translation and transcription (IVTT). Suitable methods forproducing pores are discussed in International Application Nos.PCT/GB09/001690 (published as WO 2010/004273), PCT/GB09/001679(published as WO 2010/004265) or PCT/GB10/000133 (published as WO2010/086603). Methods for inserting pores into membranes are discussedbelow.

For Exonuclease Sequencing, the transmembrane protein pore is preferablyderived from α-hemolysin (α-HL). The wild type α-HL pore is formed ofseven identical monomers or subunits (i.e. it is heptameric). Thesequence of one monomer or subunit of α-hemolysin M113R is shown in SEQID NO: 4. The transmembrane protein pore preferably comprises sevenmonomers each comprising the sequence shown in SEQ ID NO: 4 or a variantthereof. Amino acids 1, 7 to 21, 31 to 34, 45 to 51, 63 to 66, 72, 92 to97, 104 to 111, 124 to 136, 149 to 153, 160 to 164, 173 to 206, 210 to213, 217, 218, 223 to 228, 236 to 242, 262 to 265, 272 to 274, 287 to290 and 294 of SEQ ID NO: 4 form loop regions. Residues 113 and 147 ofSEQ ID NO: 4 form part of a constriction of the barrel or channel ofα-HL.

In such embodiments, a pore comprising seven proteins or monomers eachcomprising the sequence shown in SEQ ID NO: 4 or a variant thereof arepreferably used in the method of the invention. The seven proteins maybe the same (homoheptamer) or different (heteroheptamer).

A variant of SEQ ID NO: 4 is a protein that has an amino acid sequencewhich varies from that of SEQ ID NO: 4 and which retains its poreforming ability. The ability of a variant to form a pore can be assayedusing any method known in the art. For instance, the variant may beinserted into a lipid bilayer along with other appropriate subunits andits ability to oligomerise to form a pore may be determined. Methods areknown in the art for inserting subunits into membranes, such as lipidbilayers. Suitable methods are discussed above.

The variant may include modifications that facilitate covalentattachment to or interaction with a nucleic acid binding protein. Thevariant preferably comprises one or more reactive cysteine residues thatfacilitate attachment to the nucleic acid binding protein. For instance,the variant may include a cysteine at one or more of positions 8, 9, 17,18, 19, 44, 45, 50, 51, 237, 239 and 287 and/or on the amino or carboxyterminus of SEQ ID NO: 4. Preferred variants comprise a substitution ofthe residue at position 8, 9, 17, 237, 239 and 287 of SEQ ID NO: 4 withcysteine (A8C, T9C, N17C, K237C, S239C or E287C). The variant ispreferably any one of the variants described in InternationalApplication No. PCT/GB09/001,690 (published as WO 2010/004273),PCT/GB09/001,679 (published as WO 2010/004265) or PCT/GB10/000133(published as WO 2010/086603).

The variant may also include modifications that facilitate anyinteraction with nucleotides or facilitate orientation of a molecularadaptor as discussed below. The variant may also contain modificationsthat facilitate covalent attachment of a molecular adaptor.

In particular, the variant preferably contains a glutamine at position139 of SEQ ID NO: 4. The variant preferably has a cysteine at position119, 121 or 135 of SEQ ID NO: 4. A variant of SEQ ID NO: 4 may have thewild-type methionine reintroduced at position 113.

Preferred variants of SEQ ID NO: 4 have a methionine at position 113(R113M), a cysteine at position 135 (L135C) and a glutamine at position139 (N139Q). Other preferred variants of SEQ ID NO: 4 have a methionineat position 113 (R113M) and a glutamine at position 139 (N139Q). Onesuch variant is shown in SEQ ID NO: 34. A preferred transmembraneprotein pore for use in Exonuclease Sequencing comprises (a) one monomercomprising a variant of SEQ ID NO: 4 having a methionine at position 113(R113M), a cysteine at position 135 (L135C) and a glutamine at position139 (N139Q) and (b) six monomers each comprising a variant of SEQ ID NO:4 having a methionine at position 113 (R113M) and a glutamine atposition 139 (N139Q). The six monomers in (b) each preferably comprisethe sequence shown in SEQ ID NO: 34.

The variant may be a naturally occurring variant which is expressednaturally by an organism, for instance by a Staphylococcus bacterium.Alternatively, the variant may be expressed in vitro or recombinantly bya bacterium such as Escherichia coli. Variants also includenon-naturally occurring variants produced by recombinant technology.Over the entire length of the amino acid sequence of SEQ ID NO: 4, avariant will preferably be at least 50% homologous to that sequencebased on amino acid identity. More preferably, the variant polypeptidemay be at least 55%, at least 60%, at least 65%, at least 70%, at least75%, at least 80%, at least 85%, at least 90% and more preferably atleast 95%, 97% or 99% homologous based on amino acid identity to theamino acid sequence of SEQ ID NO: 4 over the entire sequence. There maybe at least 80%, for example at least 85%, 90% or 95%, amino acididentity over a stretch of 200 or more, for example 230, 250, 270 or 280or more, contiguous amino acids (“hard homology”). Homology can bedetermined as discussed above.

Amino acid substitutions may be made to the amino acid sequence of SEQID NO: 4 in addition to those discussed above, for example up to 1, 2,3, 4, 5, 10, 20 or 30 substitutions. Conservative substitutions may bemade as discussed above.

One or more amino acid residues of the amino acid sequence of SEQ ID NO:4 may additionally be deleted from the polypeptides described above. Upto 1, 2, 3, 4, 5, 10, 20 or 30 residues may be deleted, or more.

Variants may be fragments of SEQ ID NO: 4. Such fragments retainpore-forming activity. Fragments may be at least 50, 100, 200 or 250amino acids in length. A fragment preferably comprises the pore-formingdomain of SEQ ID NO: 4. Fragments typically include residues 119, 121,135. 113 and 139 of SEQ ID NO: 4.

One or more amino acids may be alternatively or additionally added tothe polypeptides described above. An extension may be provided at theamino terminus or carboxy terminus of the amino acid sequence of SEQ IDNO: 4 or a variant or fragment thereof. The extension may be quiteshort, for example from 1 to 10 amino acids in length. Alternatively,the extension may be longer, for example up to 50 or 100 amino acids. Acarrier protein may be fused to a subunit or variant.

One or more amino acids may be alternatively or additionally added tothe polypeptides described above. An extension may be provided at theamino terminus or carboxy terminus of the amino acid sequence of SEQ IDNO: 4 or a variant or fragment thereof. The extension may be quiteshort, for example from 1 to 10 amino acids in length. Alternatively,the extension may be longer, for example up to 50 or 100 amino acids. Acarrier protein may be fused to a pore or variant.

As discussed above, a variant of SEQ ID NO: 4 is a subunit that has anamino acid sequence which varies from that of SEQ ID NO: 4 and whichretains its ability to form a pore. A variant typically contains theregions of SEQ ID NO: 4 that are responsible for pore formation. Thepore forming ability of α-HL, which contains a β-barrel, is provided byβ-strands in each subunit. A variant of SEQ ID NO: 4 typically comprisesthe regions in SEQ ID NO: 4 that form β-strands. The amino acids of SEQID NO: 4 that form β-strands are discussed above. One or moremodifications can be made to the regions of SEQ ID NO: 4 that formβ-strands as long as the resulting variant retains its ability to form apore. Specific modifications that can be made to the β-strand regions ofSEQ ID NO: 4 are discussed above.

A variant of SEQ ID NO: 4 preferably includes one or more modifications,such as substitutions, additions or deletions, within its α-helicesand/or loop regions. Amino acids that form α-helices and loops arediscussed above.

The variant may be modified to assist its identification or purificationas discussed above.

A particularly preferred pore for use in Exonuclease Sequencingcomprises one subunit shown in SEQ ID NO: 36 (i.e.α-HL-E287C-QC-D5FLAGH6) and six subunits shown in SEQ ID NO: 34 (i.e.α-HL-Q).

Pores derived from α-HL can be made as discussed above with reference topores derived from Msp.

In some embodiments, the transmembrane protein pore is chemicallymodified. The pore can be chemically modified in any way and at anysite. The transmembrane protein pore is preferably chemically modifiedby attachment of a molecule to one or more cysteines (cysteine linkage),attachment of a molecule to one or more lysines, attachment of amolecule to one or more non-natural amino acids, enzyme modification ofan epitope or modification of a terminus. Suitable methods for carryingout such modifications are well-known in the art. The transmembraneprotein pore may be chemically modified by the attachment of anymolecule. For instance, the pore may be chemically modified byattachment of a dye or a fluorophore.

Any number of the monomers in the pore may be chemically modified. Oneor more, such as 2, 3, 4, 5, 6, 7, 8, 9 or 10, of the monomers ispreferably chemically modified as discussed above.

In some embodiments, the transmembrane protein pore comprises amolecular adaptor that facilitates detection of the analyte. Pores foruse in Exonuclease Sequencing typically comprise a molecular adaptor.

The molecular adaptor may directly facilitate detection of the analyteby mediating an interaction between the pore and the analyte. In suchembodiments, the presence of the adaptor improves the host-guestchemistry of the pore and the analyte and thereby improves the abilityof the pore to detect the analyte. The principles of host-guestchemistry are well-known in the art. The adaptor has an effect on thephysical or chemical properties of the pore that improves itsinteraction with the analyte. The adaptor may alter the charge of thebarrel or channel of the pore or specifically interact with or bind tothe analyte thereby facilitating its interaction with the pore.

In other embodiments, the molecular adaptor indirectly facilitatesdetection of the analyte by mediating an interaction between the poreand a product, such as a fragment, formed from processing of theanalyte. For instance, for Exonuclease Sequencing, the molecular adaptorfacilitates an interaction between the pore and individual nucleotidesdigested from the polynucleotide analyte. In such embodiments, thepresence of the adaptor improves the host-guest chemistry of the poreand the individual nucleotides and thereby improves the ability of thepore to detect the individual nucleotides. The adaptor has an effect onthe physical or chemical properties of the pore that improves itsinteraction with the individual nucleotides. The adaptor may alter thecharge of the barrel or channel of the pore or specifically interactwith or bind to the individual nucleotides thereby facilitating theirinteraction with the pore.

The molecular adaptor is preferably a cyclic molecule such as acyclodextrin, a species that is capable of hybridization, a DNA binderor interchelator, a peptide or peptide analogue, a synthetic polymer, anaromatic planar molecule, a small positively-charged molecule or a smallmolecule capable of hydrogen-bonding.

The adaptor may be cyclic. A cyclic adaptor preferably has the samesymmetry as the pore. The adaptor preferably has eight-fold symmetry ifthe pore is derived from Msp since Msp typically has eight subunitsaround a central axis. The adaptor preferably has seven-fold symmetry ifthe pore is derived from α-HL since α-HL typically has seven subunitsaround a central axis. This is discussed in more detail below.

The adaptor typically interacts with the analyte via host-guestchemistry. The adaptor is typically capable of interacting with anucleotide or polynucleotide. The adaptor comprises one or more chemicalgroups that are capable of interacting with the analyte, such as thenucleotide or polynucleotide. The one or more chemical groups preferablyinteract with the analyte, nucleotide or polynucleotide by non-covalentinteractions, such as hydrophobic interactions, hydrogen bonding, Vander Waal's forces, π-cation interactions and/or electrostatic forces.The one or more chemical groups that are capable of interacting with thenucleotide or polynucleotide are preferably positively charged. The oneor more chemical groups that are capable of interacting with thenucleotide or polynucleotide more preferably comprise amino groups. Theamino groups can be attached to primary, secondary or tertiary carbonatoms. The adaptor even more preferably comprises a ring of aminogroups, such as a ring of 6, 7 or 8 amino groups. The adaptor mostpreferably comprises a ring of seven or eight amino groups. A ring ofprotonated amino groups may interact with negatively charged phosphategroups in the nucleotide or polynucleotide.

The correct positioning of the adaptor within the pore can befacilitated by host-guest chemistry between the adaptor and the pore.The adaptor preferably comprises one or more chemical groups that arecapable of interacting with one or more amino acids in the pore. Theadaptor more preferably comprises one or more chemical groups that arecapable of interacting with one or more amino acids in the pore vianon-covalent interactions, such as hydrophobic interactions, hydrogenbonding, Van der Waal's forces, π-cation interactions and/orelectrostatic forces. The chemical groups that are capable ofinteracting with one or more amino acids in the pore are typicallyhydroxyls or amines. The hydroxyl groups can be attached to primary,secondary or tertiary carbon atoms. The hydroxyl groups may formhydrogen bonds with uncharged amino acids in the pore. Any adaptor thatfacilitates the interaction between the pore and the nucleotide orpolynucleotide can be used.

Suitable adaptors include, but are not limited to, cyclodextrins, cyclicpeptides and cucurbiturils. The adaptor is preferably a cyclodextrin ora derivative thereof. The cyclodextrin or derivative thereof may be anyof those disclosed in Eliseev, A. V., and Schneider, H-J. (1994) J. Am.Chem. Soc. 116, 6081-6088. The adaptor is more preferablyheptakis-6-amino-β-cyclodextrin (am₇-βCD),6-monodeoxy-6-monoamino-β-cyclodextrin (am₁-βCD) orheptakis-(6-deoxy-6-guanidino)-cyclodextrin (gu₇-βCD). The guanidinogroup in gu₇-βCD has a much higher pKa than the primary amines inam₇-βCD and so it more positively charged. This gu₇-βCD adaptor may beused to increase the dwell time of the nucleotide in the pore, toincrease the accuracy of the residual current measured, as well as toincrease the base detection rate at high temperatures or low dataacquisition rates.

If a succinimidyl 3-(2-pyridyldithio)propionate (SPDP) crosslinker isused as discussed in more detail below, the adaptor is preferablyheptakis(6-deoxy-6-amino)-6-N-mono(2-pyridyl)dithiopropanoyl-β-cyclodextrin(am₆amPDP₁-βCD).

More suitable adaptors include γ-cyclodextrins, which comprise 8 sugarunits (and therefore have eight-fold symmetry). The γ-cyclodextrin maycontain a linker molecule or may be modified to comprise all or more ofthe modified sugar units used in the β-cyclodextrin examples discussedabove.

The molecular adaptor is preferably covalently attached to the pore. Theadaptor can be covalently attached to the pore using any method known inthe art. The adaptor is typically attached via chemical linkage. If themolecular adaptor is attached via cysteine linkage, the one or morecysteines have preferably been introduced to the mutant by substitution.As discussed above, monomers derived from Msp can comprise a cysteineresidue at one or more of positions 88, 90, 91, 103 and 105. Eachmonomer in the pore may be chemically modified by attachment of amolecular adaptor to one or more, such as 2, 3, 4 or 5, of thesecysteines. Alternatively, the monomer may be chemically modified byattachment of a molecule to one or more cysteines introduced at otherpositions. The molecular adaptor is preferably attached to one or moreof positions 90, 91 and 103 of SEQ ID NO: 2.

For pores derived from α-HL, the correct orientation of the adaptorwithin the barrel or channel of the pore and the covalent attachment ofadaptor to the pore can be facilitated using specific modifications tothe pore. In particular, every subunit of the pore preferably has theglutamine at position 139 of SEQ ID NO: 2. One or more of the subunitsof the pore may have an arginine at position 113 of SEQ ID NO: 2. One ormore of the subunits of the pore may have a cysteine at position 119,121 or 135 of SEQ ID NO: 2 to facilitate attachment of the molecularadaptor to the pore.

The reactivity of cysteine residues may be enhanced by modification ofthe adjacent residues. For instance, the basic groups of flankingarginine, histidine or lysine residues will change the pKa of thecysteines thiol group to that of the more reactive S⁻ group. Thereactivity of cysteine residues may be protected by thiol protectivegroups such as dTNB. These may be reacted with one or more cysteineresidues of the pore before a linker is attached.

The molecule (with which the pore is chemically modified) may beattached directly to the pore or attached via a linker as disclosed inInternational Application Nos. PCT/GB09/001690 (published as WO2010/004273), PCT/GB09/001679 (published as WO 2010/004265) orPCT/GB10/000133 (published as WO 2010/086603

In a preferred embodiment, the detector comprises a polynucleotidebinding protein. This allows the method of the invention to be used tosequence polynucleotides or nucleic acids. Polynucleotides are definedbelow. Examples of polynucleotide binding proteins include, but are notlimited to, nucleic acid handling enzymes, such as nucleases,polymerases, topoisomerases, ligases and helicases, and non-catalyticbinding proteins such as those classified by SCOP (StructuralClassification of Proteins) under the Nucleic acid-binding proteinsuperfamily (50249). The polynucleotide binding protein is preferablymodified to remove and/or replace cysteine residues as described inInternational Application No. PCT/GB10/000133 (published as WO2010/086603). A preferred polynucleotide binding protein is derived fromPhi29 polymerase. The protein preferably comprises the sequence shown inSEQ ID NO: 6 or a variant thereof. This is discussed in more detailbelow. Other preferred polynucleotide binding proteins for use in theinvention include exonuclease I from E. coli (SEQ ID NO: 8), exonucleaseIII enzyme from E. coli (SEQ ID NO: 10), RecJ from T. thermophilus (SEQID NO: 12) and bacteriophage lambda exonuclease (SEQ ID NO: 14) andvariants thereof. Three identical subunits of SEQ ID NO: 14 interact toform a trimer exonuclease. The variant is preferably modified tofacilitate attachment to the membrane protein and may be any of thosediscussed in International Application No. PCT/GB09/001679 (published asWO 2010/004265) or PCT/GB10/000133 (published as WO 2010/086603). Theprotein may be any of SEQ ID NOs: 8, 10, 12, 14, 16, 18, 20, 22, 24, 26,28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48 and 50 described inInternational Application No. PCT/GB10/000,133 (published as WO2010/086603) or a variant thereof discussed in that Internationalapplication. The polynucleotide binding protein may be attached to thepore in any manner and is preferably attached as described inInternational Application No. PCT/GB09/001,679 (published as WO2010/004265) or PCT/GB10/000133 (published as WO 2010/086603).

The detector preferably comprises a polynucleotide binding protein inaddition to a transmembrane protein pore. Such detectors form modularsequencing systems that may be used in the methods of sequencing of theinvention. The polynucleotide binding protein may be attached to thepore, but does not have to be.

In Exonuclease Sequencing, the target polynucleotide is allowed tointeract with an exonuclease present in the detector. The exonuclease istypically attached to the pore in the detector. In Strand Sequencing,the detector typically comprises a polymerase in addition to the pore.The target polynucleotide is allowed to interact with the polymerase,such as Phi29 polmerase, present in the detector. The polymerase andpore are typically not attached together, but together form thedetector.

For Exonuclease Sequencing, the exonuclease is preferably covalentlyattached to the transmembrane protein pore. The exonuclease can becovalently attached to the pore using any method known in the art. Thepore and protein may be chemically fused or genetically fused. The poreand exonuclease are genetically fused if the whole construct isexpressed from a single polynucleotide sequence. Genetic fusion of apore to an exonuclease is discussed in International Application No.PCT/GB09/001679 (published as WO 2010/004265).

If the exonuclease is attached to the pore via cysteine linkage, the oneor more cysteines have preferably been introduced to the pore bysubstitution. Pores derived from Msp can of course comprise cysteineresidues at one or more of positions 10 to 15, 51 to 60, 136 to 139 and168 to 172. These positions are present in loop regions which have lowconservation amongst homologues indicating that mutations or insertionsmay be tolerated. They are therefore suitable for attaching anexonuclease. The reactivity of cysteine residues may be enhanced bymodification as described above.

The exonuclease may be attached directly to the pore or via one or morelinkers. The exonuclease may be attached to the pore using thehybridization linkers described in International Application No.PCT/GB10/000132 (published as WO 2010/086602). Alternatively, peptidelinkers may be used. Peptide linkers are amino acid sequences. Thelength, flexibility and hydrophilicity of the peptide linker aretypically designed such that it does not to disturb the functions of thepore and the exonuclease. Preferred flexible peptide linkers arestretches of 2 to 20, such as 4, 6, 8, 10 or 16, serine and/or glycineamino acids. More preferred flexible linkers include (SG)₁, (SG)₂,(SG)₃, (SG)₄, (SG)₅ and (SG)₈ wherein S is serine and G is glycine.Preferred rigid linkers are stretches of 2 to 30, such as 4, 6, 8, 16 or24, proline amino acids. More preferred rigid linkers include (P)₁₂wherein P is proline.

The detector may comprise a transmembrane protein pore chemicallymodified with a molecular adaptor and an exonuclease. Such detectors areuseful for Exonuclease Sequencing.

For Exonuclease Sequencing, the most preferred detector comprises (a) apore derived from α-HL, (b) an exonuclease covalently attached to thepore and (c) a cyclodextrin or a derivative thereof. In this preferredembodiment, the pore preferably comprises one subunit shown in SEQ IDNO: 36 (i.e. α-HL-E287C-QC-D5FLAGH6) and six subunits shown in SEQ IDNO: 34 (i.e. α-HL-Q). The exonuclease is preferably exonuclease I fromE. coli (SEQ ID NO: 8) or a variant thereof. The derivative ofcyclodextrin is preferably heptakis-6-amino-β-cyclodextrin (am₇-βCD),6-monodeoxy-6-monoamino-β-cyclodextrin (am₁-βCD) orheptakis-(6-deoxy-6-guanidino)-cyclodextrin (gu₇-βCD).

For Strand Sequencing, a preferred detector comprises (a) a pore derivedfrom Msp and (b) a Phi29 polymerase. The pore and polymerase are notattached together. This preferred embodiment is discussed in more detailbelow.

The detector may be present as an individual or single detector.Alternatively, the detector may be present in a homologous orheterologous population of two or more detectors.

Polynucleotide

A polynucleotide, such as a nucleic acid, is a macromolecule comprisingtwo or more nucleotides. The polynucleotide or nucleic acid bound by theprotein may comprise any combination of any nucleotides. The nucleotidescan be naturally occurring or artificial. The nucleotide can be oxidisedor methylated. One or more nucleotides in the polynucleotide may bedamaged. For instance, the polynucleotide may comprise a pyrimidinedimer. Such dimers are typically associated with damage by ultravioletlight and are the primary cause of skin melanomas.

A nucleotide typically contains a nucleobase, a sugar and at least onephosphate group. The nucleobase is typically heterocyclic. Nucleobasesinclude, but are not limited to, purines and pyrimidines and morespecifically adenine, guanine, thymine, uracil and cytosine. The sugaris typically a pentose sugar. Nucleotide sugars include, but are notlimited to, ribose and deoxyribose. The nucleotide is typically aribonucleotide or deoxyribonucleotide. The nucleotide typically containsa monophosphate, diphosphate or triphosphate. Phosphates may be attachedon the 5′ or 3′ side of a nucleotide.

Nucleotides include, but are not limited to, adenosine monophosphate(AMP), adenosine diphosphate (ADP), adenosine triphosphate (ATP),guanosine monophosphate (GMP), guanosine diphosphate (GDP), guanosinetriphosphate (GTP), thymidine monophosphate (TMP), thymidine diphosphate(TDP), thymidine triphosphate (TTP), uridine monophosphate (UMP),uridine diphosphate (UDP), uridine triphosphate (UTP), cytidinemonophosphate (CMP), cytidine diphosphate (CDP), cytidine triphosphate(CTP), cyclic adenosine monophosphate (cAMP), cyclic guanosinemonophosphate (cGMP), deoxyadenosine monophosphate (dAMP),deoxyadenosine diphosphate (dADP), deoxyadenosine triphosphate (dATP),deoxyguanosine monophosphate (dGMP), deoxyguanosine diphosphate (dGDP),deoxyguanosine triphosphate (dGTP), deoxythymidine monophosphate (dTMP),deoxythymidine diphosphate (dTDP), deoxythymidine triphosphate (dTTP),deoxyuridine monophosphate (dUMP), deoxyuridine diphosphate (dUDP),deoxyuridine triphosphate (dUTP), deoxycytidine monophosphate (dCMP),deoxycytidine diphosphate (dCDP) and deoxycytidine triphosphate (dCTP).The nucleotides are preferably selected from AMP, TMP, GMP, CMP, UMP,dAMP, dTMP, dGMP or dCMP.

A nucleotide may contain a sugar and at least one phosphate group (i.e.lack a nucleobase).

The nucleotides in the polynucleotide may be attached to each other inany manner. The nucleotides are typically attached by their sugar andphosphate groups as in nucleic acids. The nucleotides may be connectedvia their nucleobases as in pyrimidine dimers.

The polynucleotide may be single stranded or double stranded. At least aportion of the polynucleotide is preferably double stranded. A singlestranded polynucleotide may have one or more primers hybridised theretoand hence comprise one or more short regions of double strandedpolynucleotide. The primers may be the same type of polynucleotide asthe target polynucleotide or may be a different type of polynucleotide.

The polynucleotide can be a nucleic acid, such as deoxyribonucleic acid(DNA) or ribonucleic acid (RNA). The polynucleotide may be any syntheticnucleic acid known in the art, such as peptide nucleic acid (PNA),glycerol nucleic acid (GNA), threose nucleic acid (TNA), locked nucleicacid (LNA) or other synthetic polymers with nucleotide side chains. Thepolynucleotide bound by the protein is preferably single stranded, suchas cDNA, RNA, GNA, TNA or LNA. The polynucleotide bound by the proteinis preferably double stranded, such as DNA. Proteins that bind singlestranded polynucleotides may be used to sequence double stranded DNA aslong as the double stranded DNA is dissociated into a single strandbefore it is bound by the protein.

If the Strand Sequencing method of the invention is used thepolynucleotide analyte typically contains a portion that is doublestranded even though generally only one strand is sequenced. In aprimer/template setup, the template strand is typically sequenced (i.e.5′ threading into the pore). In any case, for Strand Sequencing, adouble stranded polynucleotide preferably comprises a single strandedleader sequence. The leader sequence can be any length, but is typically27 to 150 nucleotides in length, such as from 50 to 150 nucleotides inlength. The addition of sections of single stranded polynucleotide to adouble stranded polynucleotide can be performed in various ways. Achemical or enzymatic ligation can be done. In addition, the Nexteramethod by Epicentre is suitable. The inventors have developed a PCRmethod using a sense primer that, as usual contains a complementarysection to the start of the target region of genomic DNA, but wasadditionally preceeded with a 50 polyT section. To prevent thepolymerase from extending the complementary strand opposite the polyTsection and thereby create a blunt ended PCR product (as is normal),four abasic sites were added between the polyT section and thecomplementary priming section. These abasic sites will prevent thepolymerase from extending beyond this region and so the polyT sectionwill remain as 5′ single stranded DNA on each of the amplified copies.

Nanopore Sensing

If the detector comprises a pore, the method of the invention preferablyfurther comprises allowing the analyte to interact with the detector andmeasuring the current passing through the pore during the interactionand thereby determining the presence or absence or characteristics ofthe analyte. The analyte is present if the current flows through thepore in a manner specific for the analyte (i.e. if a distinctive currentassociated with the analyte is detected flowing through the pore). Theanalyte is absent if the current does not flow through the pore in amanner specific for the analyte. Similarly, the characteristics of theanalyte can be determined using the current flowing through the poreduring the interaction.

The invention therefore involves nanopore sensing of an analyte. Theinvention can be used to differentiate analytes of similar structure onthe basis of the different effects they have on the current passingthrough the pore. The invention can also be used to measure theconcentration of a particular analyte in a sample.

The invention may also be used in a sensor that uses many or thousandsof pores in bulk sensing applications.

The method may be carried out using any suitable membrane (such as anamphiphilic layer or a lipid bilayer) system in which a pore is insertedinto a membrane. The method is typically carried out using (i) anartificial membrane (such as an amphiphilic layer or a lipid bilayer)comprising a pore, (ii) an isolated, naturally-occurring lipid bilayercomprising a pore, or (iii) a cell having a pore inserted therein. Themethod is preferably carried out using an artificial membrane (such asan amphiphilic layer or a lipid bilayer). The membrane may compriseother transmembrane and/or intramembrane proteins as well as othermolecules in addition to the pore. Suitable apparatus and conditions arediscussed below with reference to the sequencing embodiments of theinvention. The method of the invention is typically carried out invitro.

During the interaction between the analyte and the pore, the analyteaffects the current flowing through the pore in a manner specific forthat analyte. For example, a particular analyte will reduce the currentflowing through the pore for a particular mean time period and to aparticular extent. In other words, the current flowing through the poreis distinctive for a particular analyte. Control experiments may becarried out to determine the effect a particular analyte has on thecurrent flowing through the pore. Results from carrying out the methodof the invention on a test sample can then be compared with thosederived from such a control experiment in order to identify a particularanalyte in the sample, determine whether a particular analyte is presentin the sample or determine the characteristics of the analyte. Thefrequency at which the current flowing through the pore is affected in amanner indicative of a particular analyte can be used to determine theconcentration of that analyte in the sample.

Methods of Sequencing Polynucleotides

The present invention also provides methods of estimating the sequenceof an analyte that is a target polynucleotide. The present inventionalso provides methods of sequencing an analyte that is a targetpolynucleotide. A polynucleotide is a macromolecule comprising two ormore nucleotides. The nucleotides may be any of those discussed above,including methylated, oxidised and damaged nucleotides. Thepolynucleotide may be any of those discussed above and is preferably anucleic acid.

These methods are possible because transmembrane protein pores can beused to differentiate nucleotides of similar structure on the basis ofthe different effects they have on the current passing through the pore.Individual nucleotides can be identified at the single molecule levelfrom their current amplitude when they interact with the pore. Thenucleotide is present in the pore (either individually or as part of apolynucleotide) if the current flows through the pore in a mannerspecific for the nucleotide (i.e. if a distinctive current associatedwith the nucleotide is detected flowing through the pore). Successiveidentification of the nucleotides in a target polynucleotide allows thesequence of the polynucleotide to be determined.

In one embodiment, the method comprises (a) coupling the targetpolynucleotide to a membrane; (b) allowing the target polynucleotide tointeract with a detector present in the membrane, wherein the detectorcomprises a transmembrane pore and an exonuclease, such that theexonuclease digests an individual nucleotide from one end of the targetpolynucleotide; (c) allowing the nucleotide to interact with the pore;(d) measuring the current passing through the pore during theinteraction and thereby determining the identity of the nucleotide; and(e) repeating steps (b) to (d) at the same end of the targetpolynucleotide and thereby determining the sequence of the targetpolynucleotide. In another embodiment, the method comprises (a) couplingthe target polynucleotide to a membrane; (b) allowing the targetpolynucleotide to interact with a detector present in the membrane,wherein the detector comprises a transmembrane protein pore, a molecularadaptor that facilitates an interaction between the pore and one or morenucleotides and an exonuclease, such that the exonuclease digests anindividual nucleotide from one end of the target polynucleotide; (c)allowing the nucleotide to interact with the adaptor; (d) measuring thecurrent passing through the pore during the interaction and therebydetermining the identity of the nucleotide; and (e) repeating steps (b)to (d) at the same end of the target polynucleotide and therebydetermining the sequence of the target polynucleotide. Hence, the methodinvolves nanopore sensing of a proportion of the nucleotides in a targetpolynucleotide in a successive manner in order to sequence the targetpolynucleotide. Individual nucleotides are described above and below.This is Exonuclease Sequencing.

In another embodiment, the method comprises: (a) coupling the targetpolynucleotide to a membrane; (b) allowing the target polynucleotide tointeract with a detector present in the membrane, wherein the detectorcomprises a transmembrane pore, such that the target polynucleotidemoves through the pore; and (c) measuring the current passing throughthe pore as the target polynucleotide moves with respect to the pore andthereby determining the sequence of the target polynucleotide. Inanother embodiment, the method comprises (a) coupling the targetpolynucleotide to a membrane; (b) allowing the target polynucleotide tointeract with a detector present in the membrane, wherein the detectorcomprises a transmembrane protein pore and a polynucleotide bindingprotein, preferably a polymerase, such that the protein controls themovement of the target polynucleotide through the pore and a proportionof the nucleotides in the target polynucleotide interacts with the pore;and (c) measuring the current passing through the pore during eachinteraction and thereby determining the sequence of the targetpolynucleotide. Hence, the method involves nanopore sensing of aproportion of the nucleotides in a target polynucleotide as thenucleotides individually pass through the barrel or channel in order tosequence the target polynucleotide. This is Strand Sequencing.

These methods of the invention are particularly suited for sequencingtarget polynucleotides, such as nucleic acids, because the coupling ofthe nucleic acid sequences to the membrane lowers by several orders ofmagnitude the amount of polynucleotide required. The concentrations atwhich target polynucleotides can be sequenced using the invention arediscussed above.

The whole or only part of the target polynucleotide may be sequencedusing this method. The polynucleotide can be any length. For example,the polynucleotide can be at least 10, at least 50, at least 100, atleast 150, at least 200, at least 250, at least 300, at least 400 or atleast 500 nucleotides in length. The polynucleotide can be 1000 or morenucleotides or 5000 or more nucleotides in length. The polynucleotidecan be naturally occurring or artificial. For instance, the method maybe used to verify the sequence of a manufactured oligonucleotide. Themethods are typically carried out in vitro.

The nucleotides (either digested from the target polynucleotide orpresent in the polynucleotide) may interact with the pore on either sideof the membrane. The nucleotides may interact with the pore in anymanner and at any site. As discussed above, the nucleotides preferablyreversibly bind to the pore via or in conjunction with the adaptor. Thenucleotides most preferably reversibly bind to the pore via or inconjunction with the adaptor as they pass through the pore across themembrane. The nucleotides can also reversibly bind to the barrel orchannel of the pore via or in conjunction with the adaptor as they passthrough the pore across the membrane.

During the interaction between a nucleotide and the pore, the nucleotideaffects the current flowing through the pore in a manner specific forthat nucleotide. For example, a particular nucleotide will reduce thecurrent flowing through the pore for a particular mean time period andto a particular extent. In other words, the current flowing through thepore is distinctive for a particular nucleotide. Control experiments maybe carried out to determine the effect a particular nucleotide has onthe current flowing through the pore. Results from carrying out themethod of the invention on a test sample can then be compared with thosederived from such a control experiment in order to determine thesequence of the target polynucleotide.

The sequencing methods may be carried out using any suitablemembrane/pore system in which a pore is present in or inserted into amembrane. The methods are typically carried out using a membranecomprising naturally-occurring or synthetic lipids. The membrane istypically formed in vitro. The methods are preferably not carried outusing an isolated, naturally occurring membrane comprising a pore, or acell expressing a pore. The methods are preferably carried out using anartificial membrane. The membrane may comprise other transmembraneand/or intramembrane proteins as well as other molecules in addition tothe pore.

The membrane forms a barrier to the flow of ions, nucleotides andpolynucleotides. The membrane is preferably an amphiphilic layer such asa lipid bilayer. Lipid bilayers suitable for use in accordance with theinvention are described above.

The sequencing methods of the invention are typically carried out invitro.

The sequencing methods may be carried out using any apparatus that issuitable for investigating a membrane/pore system in which a pore ispresent in or inserted into a membrane. The method may be carried outusing any apparatus that is suitable for nanopore sensing. For example,the apparatus comprises a chamber comprising an aqueous solution and abarrier that separates the chamber into two sections. The barrier has anaperture in which the membrane containing the pore is formed. Theanalyte may be coupled to the membrane in either of the two sections ofthe chamber.

The sequencing methods may be carried out using the apparatus describedin International Application No. PCT/GB08/000562.

The methods of the invention involve measuring the current passingthrough the pore during interaction with the nucleotide or as the targetpolynucleotide moves with respect to the pore. Therefore the apparatusalso comprises an electrical circuit capable of applying a potential andmeasuring an electrical signal across the membrane and pore. The methodsmay be carried out using a patch clamp or a voltage clamp. The methodspreferably involve the use of a voltage clamp.

The sequencing methods of the invention involve the measuring of acurrent passing through the pore during interaction with the nucleotideor as the target polynucleotide moves with respect to the pore. Suitableconditions for measuring ionic currents through transmembrane proteinpores are known in the art and disclosed in the Example. The method istypically carried out with a voltage applied across the membrane andpore. The voltage used is typically from −400 mV to +400 mV. The voltageused is preferably in a range having a lower limit selected from −400mV, −300 mV, −200 mV, −150 mV, −100 mV, −50 mV, −20 mV and 0 mV and anupper limit independently selected from +10 mV, +20 mV, +50 mV, +100 mV,+150 mV, +200 mV, +300 mV and +400 mV. The voltage used is morepreferably in the range 100 mV to 240 mV and most preferably in therange of 160 mV to 240 mV. It is possible to increase discriminationbetween different nucleotides by a pore by using an increased appliedpotential.

The sequencing methods are typically carried out in the presence of anyalkali metal chloride salt. In the exemplary apparatus discussed above,the salt is present in the aqueous solution in the chamber. Potassiumchloride (KCl), sodium chloride (NaCl) or caesium chloride (CsCl) istypically used. KCl is preferred. The salt concentration is typicallyfrom 0.1 to 2.5M, from 0.3 to 1.9M, from 0.5 to 1.8M, from 0.7 to 1.7M,from 0.9 to 1.6M or from 1M to 1.4M. The salt concentration ispreferably from 150 mM to 1M. High salt concentrations provide a highsignal to noise ratio and allow for currents indicative of the presenceof a nucleotide to be identified against the background of normalcurrent fluctuations. Lower salt concentrations may be used ifnucleotide detection is carried out in the presence of an enzyme, suchas when sequencing polynucleotides. This is discussed in more detailbelow.

The methods are typically carried out in the presence of a buffer. Inthe exemplary apparatus discussed above, the buffer is present in theaqueous solution in the chamber. Any buffer may be used in the method ofthe invention. One suitable buffer is Tris-HCl buffer. The methods aretypically carried out at a pH of from 4.0 to 12.0, from 4.5 to 10.0,from 5.0 to 9.0, from 5.5 to 8.8, from 6.0 to 8.7 or from 7.0 to 8.8 or7.5 to 8.5. The pH used is preferably about 7.5.

The methods are typically carried out at from 0° C. to 100° C., from 15°C. to 95° C., from 16° C. to 90° C., from 17° C. to 85° C., from 18° C.to 80° C., 19° C. to 70° C., or from 20° C. to 60° C. The methods may becarried out at room temperature. The methods are preferably carried outat a temperature that supports enzyme function, such as about 37° C.

As mentioned above, good nucleotide discrimination can be achieved atlow salt concentrations if the temperature is increased. In addition toincreasing the solution temperature, there are a number of otherstrategies that can be employed to increase the conductance of thesolution, while maintaining conditions that are suitable for enzymeactivity. One such strategy is to use the lipid bilayer to divide twodifferent concentrations of salt solution, a low salt concentration ofsalt on the enzyme side and a higher concentration on the opposite side.One example of this approach is to use 200 mM of KCl on the cis side ofthe membrane and 500 mM KCl in the trans chamber. At these conditions,the conductance through the pore is expected to be roughly equivalent to400 mM KCl under normal conditions, and the enzyme only experiences 200mM if placed on the cis side. Another possible benefit of usingasymmetric salt conditions is the osmotic gradient induced across thepore. This net flow of water could be used to pull nucleotides into thepore for detection. A similar effect can be achieved using a neutralosmolyte, such as sucrose, glycerol or PEG. Another possibility is touse a solution with relatively low levels of KCl and rely on anadditional charge carrying species that is less disruptive to enzymeactivity.

The target polynucleotide being analysed can be combined with knownprotecting chemistries to protect the polynucleotide from being actedupon by the binding protein or exonuclease while in the bulk solution.The pore can then be used to remove the protecting chemistry. This canbe achieved either by using protecting groups that are unhybridised bythe pore, binding protein or enzyme under an applied potential (WO2008/124107) or by using protecting chemistries that are removed by thebinding protein or enzyme when held in close proximity to the pore (J AmChem. Soc. 2010 Dec. 22; 132(50):17961-72).

Exonuclease Sequencing

In one embodiment, the method of sequencing an analyte which is a targetpolynucleotide involves allowing the target polynucleotide to interactwith an exonuclease enzyme. Any of the exonuclease enzymes discussedabove may be used in the method. The exonuclease releases individualnucleotides from one end of the target polynucleotide. The enzyme may becovalently attached to the pore as discussed above.

An individual nucleotide is a single nucleotide. An individualnucleotide is one which is not bound to another nucleotide orpolynucleotide by a nucleotide bond. A nucleotide bond involves one ofthe phosphate groups of a nucleotide being bound to the sugar group ofanother nucleotide. An individual nucleotide is typically one which isnot bound by a nucleotide bond to another polynucleotide of at least 5,at least 10, at least 20, at least 50, at least 100, at least 200, atleast 500, at least 1000 or at least 5000 nucleotides. For example, theindividual nucleotide has been digested from a target polynucleotide,such as a DNA or RNA strand. The individual nucleotide may be any ofthose discussed above.

Exonucleases are enzymes that typically latch onto one end of apolynucleotide and digest the polynucleotide one nucleotide at a timefrom that end. The exonuclease can digest the polynucleotide in the 5′to 3′ direction or 3′ to 5′ direction. The end of the polynucleotide towhich the exonuclease binds is typically determined through the choiceof enzyme used and/or using methods known in the art. Hydroxyl groups orcap structures at either end of the polynucleotide may typically be usedto prevent or facilitate the binding of the exonuclease to a particularend of the polynucleotide.

The method involves allowing the polynucleotide to interact with theexonuclease so that the nucleotides are digested from the end of thepolynucleotide at a rate that allows identification of a proportion ofnucleotides as discussed above. Methods for doing this are well known inthe art. For example, Edman degradation is used to successively digestsingle amino acids from the end of polypeptide such that they may beidentified using High Performance Liquid Chromatography (HPLC). Ahomologous method may be used in the present invention.

The rate at which the exonuclease functions is typically slower than theoptimal rate of a wild-type exonuclease. A suitable rate of activity ofthe exonuclease in the method of sequencing involves digestion of from0.5 to 1000 nucleotides per second, from 0.6 to 500 nucleotides persecond, 0.7 to 200 nucleotides per second, from 0.8 to 100 nucleotidesper second, from 0.9 to 50 nucleotides per second or 1 to 20 or 10nucleotides per second. The rate is preferably 1, 10, 100, 500 or 1000nucleotides per second. A suitable rate of exonuclease activity can beachieved in various ways. For example, variant exonucleases with areduced optimal rate of activity may be used in accordance with theinvention.

The Exonuclease Sequencing methods of the invention have additionaladvantages beyond the reduction in the amount of polynucleotide needed.The inventors have studied the presentation of single stranded DNA insolution to an Exonuclease-Nanopore (“X-Pore”)/membrane system underpotential. When DNA analyte is introduced into the system, the pore maybecome blocked permanently or temporarily, preventing the detection ofindividual nucleotides. When one end of the DNA analyte is localisedaway from the pore, for example by coupling to the membrane,surprisingly it was found that this blocking is no longer observed. Italso increases the number of potential DNA threading events for theenzyme due to the increased effective concentration of being in the sameplane as the analyte. This acts to lower the binding time betweenanalytes and increase sequencing throughput.

Strand Sequencing

Strand Sequencing involves the controlled and stepwise translocation ofpolynucleotides through a pore. A polynucleotide is a macromoleculecomprising two or more nucleotides. The polynucleotide bound by theprotein may comprise any combination of any nucleotides. The nucleotidesmay be any of those discussed above.

The Strand Sequencing method of the invention typically uses apolynucleotide binding protein to control the movement of the targetpolynucleotide through the pore. Examples of such proteins are givenabove. The polynucleotide binding protein is preferably a polynucleotidehandling enzyme. A polynucleotide handling enzyme is a polypeptide thatis capable of interacting with and modifying at least one property of apolynucleotide. The enzyme may modify the polynucleotide by cleaving itto form individual nucleotides or shorter chains of nucleotides, such asdi- or trinucleotides. The enzyme may modify the polynucleotide byorienting it or moving it to a specific position. The polynucleotidehandling enzyme does not need to display enzymatic activity as long asit is capable of binding the target polynucleotide and controlling itsmovement through the pore. For instance, the enzyme may be modified toremove its enzymatic activity or may be used under conditions whichprevent it from acting as an enzyme. Such conditions are discussed inmore detail below.

The polynucleotide handling enzyme is preferably derived from anucleolytic enzyme. The polynucleotide handling enzyme used in theconstruct of the enzyme is more preferably derived from a member of anyof the Enzyme Classification (EC) groups 3.1.11, 3.1.13, 3.1.14, 3.1.15,3.1.16, 3.1.21, 3.1.22, 3.1.25, 3.1.26, 3.1.27, 3.1.30 and 3.1.31. Theenzyme may be any of those disclosed in International Application No.PCT/GB10/000133 (published as WO 2010/086603).

Preferred enzymes are polymerases, exonucleases, helicases, translocasesand topoisomerases, such as gyrases. Suitable enzymes include, but arenot limited to, exonuclease I from E. coli (SEQ ID NO: 8), exonucleaseIII enzyme from E. coli (SEQ ID NO: 10), RecJ from T. thermophilus (SEQID NO: 12) and bacteriophage lambda exonuclease (SEQ ID NO: 14) andvariants thereof. Three subunits comprising the sequence shown in SEQ IDNO: 14 or a variant thereof interact to form a trimer exonuclease. Theenzyme is most preferably derived from Phi29 DNA polymerase. An enzymederived from Phi29 polymerase comprises the sequence shown in SEQ ID NO:6 or a variant thereof.

According to one embodiment the polynucleotide binding protein iscoupled or tethered to the membrane and is able both to bind to analytepolynucleotide and then to control translocation of the analyte throughthe pore. In this embodiment, the analyte polynucleotide may be coupledto the membrane via the polynucleotide binding protein. The analytepolynucleotide and the polynucleotide binding protein may both becoupled to the membrane, preferably by different coupling methods. Thepolynucleotide binding protein is preferably a helicase.

A variant of SEQ ID NOs: 6, 8, 10, 12 or 14 is an enzyme that has anamino acid sequence which varies from that of SEQ ID NO: 6, 8, 10, 12 or14 and which retains polynucleotide binding ability. The variant mayinclude modifications that facilitate binding of the polynucleotideand/or facilitate its activity at high salt concentrations and/or roomtemperature.

Over the entire length of the amino acid sequence of SEQ ID NO: 6, 8,10, 12 or 14, a variant will preferably be at least 50% homologous tothat sequence based on amino acid identity. More preferably, the variantpolypeptide may be at least 55%, at least 60%, at least 65%, at least70%, at least 75%, at least 80%, at least 85%, at least 90% and morepreferably at least 95%, 97% or 99% homologous based on amino acididentity to the amino acid sequence of SEQ ID NO: 6, 8, 10, 12 or 14over the entire sequence. There may be at least 80%, for example atleast 85%, 90% or 95%, amino acid identity over a stretch of 200 ormore, for example 230, 250, 270 or 280 or more, contiguous amino acids(“hard homology”). Homology is determined as described above. Thevariant may differ from the wild-type sequence in any of the waysdiscussed above with reference to SEQ ID NO: 2. The enzyme may becovalently attached to the pore as discussed above.

The enzyme is not required to be in as close a proximity to the porelumen as for individual nucleotide sequencing as there is no potentialfor disorder in the series in which nucleotides reach the sensing moietyof the pore.

The two strategies for strand DNA sequencing are the translocation ofthe DNA through the nanopore, both cis to trans and trans to cis, eitherwith or against an applied potential. One of the most advantageousmechanisms for strand sequencing is the controlled translocation ofsingle strand DNA through the nanopore under an applied potential.Exonucleases that act progressively or processively on double strandedDNA can be used on the cis side of the pore to feed the remaining singlestrand through under an applied potential or the trans side under areverse potential. Likewise, a helicase that unwinds the double strandedDNA can also be used in a similar manner. There are also possibilitiesfor sequencing applications that require strand translocation against anapplied potential, but the DNA must be first “caught” by the enzymeunder a reverse or no potential. With the potential then switched backfollowing binding the strand will pass cis to trans through the pore andbe held in an extended conformation by the current flow. The singlestrand DNA exonucleases or single strand DNA dependent polymerases canact as molecular motors to pull the recently translocated single strandback through the pore in a controlled stepwise manner, trans to cis,against the applied potential. Alternatively, the single strand DNAdependent polymerases can act as molecular brake slowing down themovement of a polynucleotide through the pore.

In the most preferred embodiment, Strand Sequencing is carried out usinga pore derived from Msp and a Phi29 DNA polymerase. The method maycomprise (a) coupling the target polynucleotide to a membrane; (b)allowing the target polynucleotide to interact with a detector in themembrane, which detector comprises a pore derived from Msp and a Phi29DNA polymerase, such that the polymerase controls the movement of thetarget polynucleotide through the pore; and (c) measuring the currentpassing through the pore as the target polynucleotide moves with respectto the pore and thereby determining the sequence of the targetpolynucleotide, wherein steps (b) and (c) are carried out with a voltageapplied across the pore. The method may comprise (a) coupling the targetpolynucleotide to a membrane; (b) allowing the target polynucleotide tointeract with a detector in the membrane, which detector comprises apore derived from Msp and a Phi29 DNA polymerase, such that thepolymerase controls the movement of the target polynucleotide throughthe pore and a proportion of the nucleotides in the targetpolynucleotide interacts with the pore; and (c) measuring the currentpassing through the pore during each interaction and thereby determiningthe sequence of the target polynucleotide, wherein steps (b) and (c) arecarried out with a voltage applied across the pore. When the targetpolynucleotide is contacted with a Phi29 DNA polymerase and a porederived from Msp, the target polynucleotide firstly forms a complex withthe Phi29 DNA polymerase. When the voltage is applied across the pore,the target polynucleotide/Phi29 DNA polymerase complex forms a complexwith the pore and controls the movement of the target polynucleotidethrough the pore.

These Msp/Phi29 embodiments have three unexpected advantages. First, thetarget polynucleotide moves through the pore at a rate that iscommercially viable yet allows effective sequencing. The targetpolynucleotide moves through the Msp pore more quickly than it doesthrough a hemolysin pore. Second, an increased current range is observedas the polynucleotide moves through the pore allowing the sequence to bedetermined more easily. Third, a decreased current variance is observedwhen the specific pore and polymerase are used together therebyincreasing the signal-to-noise ratio.

Any polynucleotide described above may be sequenced. At least a portionof the polynucleotide is preferably double stranded.

The pore may be any of the pores discussed above. The pore may compriseeight monomers comprising the sequence shown in SEQ ID NO: 2 or avariant thereof.

Wild-type Phi29 DNA polymerase has polymerase and exonuclease activity.It may also unzip double stranded polynucleotides under the correctconditions. Hence, the enzyme may work in three modes. This is discussedin more detail below.

The Phi29 DNA polymerase may comprise the sequence shown in SEQ ID NO: 6or a variant thereof. A variant of SEQ ID NOs: 6 is an enzyme that hasan amino acid sequence which varies from that of SEQ ID NO: 6 and whichretains polynucleotide binding activity. The variant must work in atleast one of the three modes discussed below. Preferably, the variantworks in all three modes. The variant may include modifications thatfacilitate handling of the polynucleotide and/or facilitate its activityat high salt concentrations and/or room temperature.

Over the entire length of the amino acid sequence of SEQ ID NO: 6, avariant will preferably be at least 40% homologous to that sequencebased on amino acid identity. More preferably, the variant polypeptidemay be at least 50%, at least 55%, at least 60%, at least 65%, at least70%, at least 75%, at least 80%, at least 85%, at least 90% and morepreferably at least 95%, 97% or 99% homologous based on amino acididentity to the amino acid sequence of SEQ ID NO: 4 over the entiresequence. There may be at least 80%, for example at least 85%, 90% or95%, amino acid identity over a stretch of 200 or more, for example 230,250, 270 or 280 or more, contiguous amino acids (“hard homology”).Homology is determined as described above. The variant may differ fromthe wild-type sequence in any of the ways discussed above with referenceto SEQ ID NO: 2. The enzyme may be covalently attached to the pore asdiscussed above.

Any of the systems, apparatus or conditions discussed above may be usedin accordance with this preferred embodiment. The salt concentration istypically from 0.15M to 0.6M. The salt is preferably KCl.

The method may be carried out in one of three preferred ways based onthe three modes of the Phi29 DNA polymerase. Each way includes a methodof proof-reading the sequence. First, the method is preferably carriedout using the Phi29 DNA polymerase as a polymerase. In this embodiment,steps (b) and (c) are carried out in the presence of free nucleotidesand an enzyme cofactor such that the polymerase moves the targetpolynucleotide through the pore against the field resulting from theapplied voltage. The target polynucleotide moves in the 5′ to 3′direction. The free nucleotides may be one or more of any of theindividual nucleotides discussed above. The enzyme cofactor is a factorthat allows the Phi29 DNA polymerase to function either as a polymeraseor an exonuclease. The enzyme cofactor is preferably a divalent metalcation. The divalent metal cation is preferably Mg²⁺, Mn²⁺, Ca²⁺ orCo²⁺. The enzyme cofactor is most preferably Mg²⁺. The method preferablyfurther comprises (d) removing the free nucleotides such that thepolymerase moves the target polynucleotide through the pore with thefield resulting from the applied voltage (i.e. in the 3′ and 5′direction) and a proportion of the nucleotides in the targetpolynucleotide interacts with the pore and (e) measuring the currentpassing through the pore during each interaction and thereby proofreading the sequence of the target polynucleotide obtained in step (c),wherein steps (d) and (e) are also carried out with a voltage appliedacross the pore.

Second, the method is preferably carried out using the Phi29 DNApolymerase as an exonuclease. In this embodiment, wherein steps (b) and(c) are carried out in the absence of free nucleotides and the presenceof an enzyme cofactor such that the polymerase moves the targetpolynucleotide through the pore with the field resulting from theapplied voltage. The target polynucleotide moves in the 3′ to 5′direction. The method preferably further comprises (d) adding freenucleotides such that the polymerase moves the target polynucleotidethrough the pore against the field resulting from the applied voltage(i.e. in the 5′ to 3′ direction) and a proportion of the nucleotides inthe target polynucleotide interacts with the pore and (e) measuring thecurrent passing through the pore during each interaction and therebyproof reading the sequence of the target polynucleotide obtained in step(c), wherein steps (d) and (e) are also carried out with a voltageapplied across the pore.

Third, the method is preferably carried out using the Phi29 DNApolymerase in unzipping mode. In this embodiment, steps (b) and (c) arecarried out in the absence of free nucleotides and the absence of anenzyme cofactor such that the polymerase controls the movement of thetarget polynucleotide through the pore with the field resulting from theapplied voltage (as it is unzipped). In this embodiment, the polymeraseacts like a brake preventing the target polynucleotide from movingthrough the pore too quickly under the influence of the applied voltage.The method preferably further comprises (d) lowering the voltage appliedacross the pore such that the target polynucleotide moves through thepore in the opposite direction to that in steps (b) and (c) (i.e. as itre-anneals) and a proportion of the nucleotides in the targetpolynucleotide interacts with the pore and (e) measuring the currentpassing through the pore during each interaction and thereby proofreading the sequence of the target polynucleotide obtained in step (c),wherein steps (d) and (e) are also carried out with a voltage appliedacross the pore.

Kits

The present invention also provides kits for sequencing an analyte whichis a target polynucleotide. The kit comprises (a) a transmembrane pore,such as a transmembrane protein pore, (b) a polynucleotide bindingprotein and (c) means to couple the target polynucleotide to a membrane.In a preferred embodiment, the polynucleotide binding protein is anexonuclease and the kit further comprises a molecular adaptor thatfacilitates an interaction between the pore and one or more nucleotidesin the target polynucleotide. Such a kit may be used for ExonucleaseSequencing. In another preferred embodiment, the kit comprises thecomponents of a membrane, such as the phospho lipids needed to form alipid bilayer.

The means to couple the target polynucleotide to a membrane preferablycomprises a reactive group. Suitable groups include, but are not limitedto, thiol, cholesterol, lipid and biotin groups. Any of the embodimentsdiscussed above with reference to the methods of the invention areequally applicable to the kits of the invention.

The kits of the invention may additionally comprise one or more otherreagents or instruments which enable any of the embodiments mentionedabove to be carried out. Such reagents or instruments include one ormore of the following: suitable buffer(s) (aqueous solutions), means toobtain a sample from a subject (such as a vessel or an instrumentcomprising a needle), means to amplify and/or express polynucleotides, amembrane as defined above or voltage or patch clamp apparatus. Reagentsmay be present in the kit in a dry state such that a fluid sampleresuspends the reagents. The kit may also, optionally, compriseinstructions to enable the kit to be used in the method of the inventionor details regarding which patients the method may be used for. The kitmay, optionally, comprise nucleotides.

Apparatus

The invention also provides an apparatus for sequencing an analyte whichis a target polynucleotide. The apparatus comprises (a) a membrane, (b)a plurality of transmembrane pores in the membrane, (c) a plurality ofpolynucleotide binding proteins and (d) a plurality of targetpolynucleotides coupled to the membrane. The plurality of polynucleotidebinding proteins may be in the membrane. The apparatus may be anyconventional apparatus for analyte analysis, such as an array or a chip.In a preferred embodiment, the polynucleotide binding protein is anexonuclease and the apparatus comprises a molecular adaptor thatfacilitates an interaction between the pore and one or more nucleotidesin the target polynucleotide. Such an apparatus may be used forExonuclease Sequencing. Any of the embodiments discussed above withreference to the methods of the invention are equally applicable to thekits of the invention. The apparatus preferably comprises:

a sensor device that is capable of supporting the membrane and pluralityof pores and being operable to perform polynucleotide sequencing usingthe pores and proteins; and

at least one reservoir for holding material for performing thesequencing.

The apparatus preferably comprises:

a sensor device that is capable of supporting the membrane and pluralityof pores and being operable to perform polynucleotide sequencing usingthe pores and proteins;

at least one reservoir for holding material for performing thesequencing;

a fluidics system configured to controllably supply material from the atleast one reservoir to the sensor device; and

one or more, such as a plurality, of containers for receiving respectivesamples, the fluidics system being configured to supply the samplesselectively from the one or more containers to the sensor device. Theapparatus may be any of those described in International Application No.PCT/GB08/004127 (published as WO 2009/077734), PCT/GB10/000789(published as WO 2010/122293), International Application No.PCT/GB10/002206 (not yet published) or International Application No.PCT/US99/25679 (published as WO 00/28312).

The following Examples illustrate the invention:

1. Example 1—Exonuclease Sequencing

1.1 Materials and Methods

1.1.1 Materials and Oligonucleotides

Oligonucleotides were purchased from either ADTBio or IDTDNA. Details ofthe exact sequences and modifications can be found in the Table below(SEQ ID NOs 18 to 21).

Modification   In- SEQ Sup- ter- Length ID plier Name 5′ nal 3′ (nt)Sequence NO: Code ONLA Chol- — — 70 TTTTTTTTT 18 ATDBio 0692 TEGTTTTTTTTT A8691 TTTTTTTTT TTTTTTTTT TTTTTTTTT TTTTTTTTT TTTTTTTTTTTTTTTT ONLA Chol- — — 70 AAAAAAAAA 19 ATDBio 0682 TEG AAAAAAAAA A887AAAAAAAAA AAAAAAAAA AAAAAAAAA AAAAAAAAA AAAAAAAAA AAAAAAA ONLA Chol- — —70 CCCCCCCCC 20 ATDBio 0683 TEG CCCCCCCCC A8874 CCCCCCCCC CCCCCCCCCCCCCCCCCC CCCCCCCCC CCCCCCCCC CCCCCCC ONLA Strep- — — 70 TTTTTTTTT 18IDT 0693 Btn: TTTTTTTTT 60739014 ssDNA TTTTTTTTT TTTTTTTTT TTTTTTTTTTTTTTTTTT TTTTTTTTT TTTTTTT ONLA Strep- — — 70 TGTGTTCTA 21 IDT 0694Btn: TGTCTTATT 60739013 ssDNA CTTACTTCG TTATTCTTG TCTCTATTC TGTTTATGTTTCTTGTTT GTTAGCA ONLA — — — 70 TTTTTTTTT 18 IDT 0706 TTTTTTTTT 60692267TTTTTTTTT TTTTTTTTT TTTTTTTTT TTTTTTTTT TTTTTTTTT TTTTTTT

Recombinant Streptavidin, expressed in E. coli, was purchased from SigmaAldrich (S0677). The synthetic lipids1,2-diphytanoyl-sn-glycero-3-phosphocholine (16:0 4ME PC) and1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-cap biotinyl (16:0Cap Biotinyl PE) were purchased from Avanti Polar Lipids.

1.1.2 HPLC Purification of Mono-Substituted Streptavidin

1 uM of 5′-biotin modified DNA was mixed with 10 uM of streptavidin in25 mM Tris.HCl, 400 mM KCl, 10 mM MgCl2, pH 7.5 and incubated for 30mins at 22° C. Mono-substituted Strep:DNA conjugates were separatedusing an Agilent 1200 analytical LC system comprising a binary pump,column oven maintained at 23° C., UV detector with 13 ul flow cell, withboth sample compartment and fractions maintained at 4° C. The column wasan Agilent BioMonolith QA run at 1 ml min⁻¹, and samples were separatedon a gradient from 30 mM-1.1 M NaCl in 100 mM Tris pH 8.5.Quantification of purified mono-substituted Strep:DNA conjugates wascarried out using densitometry following gel electrophoresis using aseries of DNA standards to create a standard curve.

1.1.3 Single Channel Recordings from Planar Lipid Bilayers

Bilayers were formed by apposition of two monolayers of either 100% 16:04ME PC or 95% 16:0 4ME PC, 5% 16:0 Cap Biotinyl PE. Bilayers were formedacross a 60-150 μm diameter aperture in Teflon film (25 μm thicknessfrom Goodfellow, Malvern, Pa.), which divided a chamber into two buffercompartments (cis and trans) each with a volume of 1 ml. Bilayers wereformed across the aperture by consecutively raising the buffer level ineach compartment until a high resistance seal was observed (≥10 GΩ).Unless otherwise stated, DNA and protein were added to the ciscompartment, which was connected to ground. No reagents were added tothe trans compartment, which was connected to the head-stage of theamplifier. Unless stated otherwise, experiments were carried out in 25mM Tris.HCl, 400 mM KCl, 10 mM MgCl2, pH 7.5, at 22° C.

1.2 Results

1.2.1 Single Molecule Detection of Tethered Analytes

Nanopore detection rates for single stranded DNA free in solution can bedetermined by measuring the number of DNA translocations (events)through the nanopore per second. A DNA translocation can be identifiedby a signature transient current blockade in the digital recording. Fortethered analytes the number of interactions can similarly be calculatedprovided the DNA is only transiently tethered to the bilayer, such asvia a cholesterol group. As the free end of the DNA enters the nanoporeit will reside in the barrel until the tethered end becomes free of thebilayer and so the molecule can translocate (FIGS. 2A and 2B). If thetethering is more stable then the block will be permanent (FIGS. 2C and2D).

A 50% mix of cholesterol modified PolyA and PolyC (ONLA0682 and ONLA0683respectively) were assayed at 10, 100 and 1000 pM final concentration toestablish the effect of Chol-DNA on the event rate and dwell time (Tablebelow). This was compared to the event rate for free single strandedDNA.

Free Analyte Tethered Analyte 100 nM 10 pM 100 pM 1000 pM Event Rate s⁻¹120 mV 0.01 0.015 0.045 2.5 160 mV 0.74 0.15 1.05 26

Rates increase with voltage at all concentrations (Table above) and ofcourse event rates are higher at higher concentrations. At the lowerconcentrations and voltages the event rates are too low to really beconsidered meaningful. That is, it is likely that most, if not all, ofthe events at those conditions are just the occasional false-positive.It is somewhat surprising however that at higher current levels asignificant number of DNA events are seen with only 10 pM of DNA. Inspite of the DNA concentration being at least 100 times lower, the eventrates are much higher with cholesterol modified DNA. It is somewhatsurprising that at the higher current levels (≥160 mV) a significantnumber of DNA events are still seen with only 10 pM of DNA and these DNAevents occur at a frequency similar to 100 nM of unmodified ssDNA. Itcan be estimated that tethering of the DNA improves the detection of theDNA analyte by 3-4 orders of magnitude. For certain applications thetransient nature of the tethering might be preferred. If a stabletethering molecule were attached directly to either the 5′ or 3′-end ofthe DNA then some sequence data will be lost as the sequencing runcannot continue to the end of the DNA, due to the distance between thebilayer and the enzymes active site. If the tethering is more transientthen when the tethered end randomly becomes free of the bilayer then theenzyme can process the DNA to completion.

1.3 Conclusions

We have demonstrated here the potential to improve the detectionefficiency of a nanopore detector for an analyte by approximately 3-4orders of magnitude. The rapid pore blocking suggests that this tetheredanalyte is still available for proteins both from solution and in thebilayer (such as an enzyme or a nanopore construct respectively) and sohas the potential as either a delivery mechanism to the pore itself orto a nanopore-enzyme construct.

Various means of analyte attachment to the lipid bilayer are availableand most have been reported for the tethering of ssDNA, as functionalchemistry can be easily incorporated during oligonucleotide synthesis.In the preferred means a ddNTP modified with a biotin can beincorporated to the 3′-end of ssDNA using terminal transferase. Bymixing with streptavidin the analyte DNA can then be added to a singlepore in a lipid bilayer containing 1-5% Biotin PE where it will becometethered. Alternatively if the sequence is known then the DNA can behybridised to complementary synthetic DNA already modified at one end tobe lipophilic.

Another advantage of tethering the analyte is that you have control overone end of the DNA. It can be seen above that DNA will rapidly block thepore if one end is held in close proximity, in the above case this isthe bilayer. If a DNA handling enzyme, such as an exonuclease, isattached to the nanopore then it will bind one end of the DNA and againlocalise it to the pore and so the other end will rapidly block. If DNAis immobilised however then when the enzyme binds to one end then bothare now occupied and unavailable for the pore.

The need for low analyte requirement DNA sequencing is for applicationssuch as single cell sequencing for epigenetics and also screening fromlow volume biological samples. The current Illumina Genome analysersystem requires 100 ng to 1 ug DNA for a sequencing library prep. Asingle 128 channel chip for nanopore sequencing could use ˜0.5 ng DNAwithout the need for amplification; based on 1000mer fragment generationand read length at 10 pM concentration.

2. Example 2—Strand Sequencing

In addition to the work for attaching ssDNA to the lipid membrane forExonuclease Sequencing, the technique can also be adapted to a StrandSequencing approach. In Strand Sequencing, a portion of a polynucleotidestrand is threaded through the nanopore under an applied potential. Thestrand is typically DNA or RNA, for example single stranded or doublestranded DNA. Preferably the strand is single stranded DNA (ssDNA). Thebase residues comprised in the strand interact with the nanopore and asignal is generated that is characteristic of each residue. The strandis moved through the pore, causing variation to the signal. The signalcan be used to infer the sequence of the DNA strand.

One embodiment of Strand Sequencing uses a protein pore embedded in alipid membrane. Electrodes are placed either side of the lipid membranein an electrolyte and a potential is applied across the system. Underthe potential the polynucleotide translocates the pore. The currentthrough the protein pore can be measured and used to recognise bases asthey pass through the trans-membrane barrel of the pore. Typically theprotein pore will be a bacterial membrane protein, such as a porin or atoxin. Preferably the pore is a hemolysin, a gramicidin or an MspA.

The rate that DNA translocates through a pore may be too fast to allowaccurate identification of each base, therefore it may be desirable toslow the translocation. One method for slowing the translocation of aDNA strand is to use a DNA handling protein, such as a DNA polymerase.The DNA handling protein may be attached to the pore, for example bycovalent bonding, either directly or via linker groups. Typically theDNA handling protein is attached to the pore for exonuclease sequencingapplications. Commonly for strand sequencing applications the DNAhandling protein is not attached to the pore.

For a Strand Sequencing approach, it is desirable to have a DNA handlingprotein that has a very long binding time on top of the nanopore. A longbinding time allows for many nucleotides to be processed through the DNAhandling protein and thus through the nanopore. For a polymerase, atypical rate of processing may be around 20 nucleotides a second. Abinding time of 10 minutes would allow the movement of 12,000nucleotides. A binding time of one minute would allow 120 nucleotides tobe processed.

Using this approach, a long binding time is also related to the readlength. Currently, a read length of around 100 nucleotides would besufficient to rival existing technologies, although longer read lengthsare desirable, for example a read length of 200, 500 or 1000nucleotides. Preferred read lengths are at least 5000 nucleotides, morepreferably 10000 or 50000 nucleotides. One advantage of a long readlength is that it greatly reduces the complexity of the bioinformaticsneeded to analyse sequencing information.

Typically a DNA handling protein is a DNA polymerase. Preferred DNAhandling proteins include Phi29 DNA polymerase.

2.1 Materials and Methods

Bilayers were formed by apposition of two monolayers of either 100%DPhPC or 99% DPhPC, 1% 16:0 Cap Biotinyl PE. Bilayers were formed acrossa 60-150 μm diameter aperture in Teflon film (25 μm thickness fromGoodfellow, Malvern, Pa.), which divided a chamber into two buffercompartments (cis and trans) each with a volume of 1 ml. Bilayers wereformed across the aperture by consecutively raising the buffer level ineach compartment until a high resistance seal was observed (≥10 GΩ).Unless otherwise stated, DNA and protein were added to the ciscompartment, which was connected to ground. No reagents were added tothe trans compartment, which was connected to the head-stage of theamplifier. Experiments were carried out with 400 mM KCl, 25 mM Tris.HCl,10 uM EDTA, pH 7.5. The hemolysin mutant used was HL-(E111N/K147N)7 (SEQID NO: 38).

1 uM of 5′-biotin modified DNA (StrandDNA1) was mixed with 10 uM ofstreptavidin in 25 mM Tris.HCl, 400 mM KCl, 10 mM MgCl2, pH 7.5 andincubated for 30 mins at 22° C. Mono-substituted Strep:DNA conjugateswere separated using an Agilent 1200 analytical LC system comprising abinary pump, column oven maintained at 23° C., UV detector with 13 ulflow cell, with both sample compartment and fractions maintained at 4°C. The column was an Agilent BioMonolith QA run at 1 ml min-1, andsamples were separated on a gradient from 30 mM-1.1 M NaCl in 100 mMTris pH 8.5. Quantification of purified mono-substituted Strep:DNAconjugates was carried out using densitometry following gelelectrophoresis using a series of DNA standards to create a standardcurve. To form StrandDNA3, the DNA-streptavidin complex was hybrisidedwith a 5× excess of StrandDNA2 bp heating to 50° C. for 10 minutes on aPCR heating block. The temperature was reduced to 23° C. at a rate of 2degrees a minute.

For membrane tethering runs, the bilayer was formed with 99% DPhPC, 1%16:0 Cap Biotinyl PE. Once the bilayer was formed, 1 nM of StrandDNA3was added to the cis chamber and mixed well. A control section wasrecorded for 5 minutes at +180 mV to obtain DNA binding events to thenanopore. After the control section was recorded, 5 nM of KF (exo minus)(NEB) was added and the signal was recorded for 5 minutes at +180 mV.

For runs where the analyte is in solution, the bilayer was formed with100% DPhPC. StrandDNA6 was produced by hybridising StrandDNA4 andStrandDNA5 at equimolar concentrations. Hybridisation was performed byheating to 50° C. on a PCR block for 10 minutes, then cooling to 23° C.at 2° C./min. Once the bilayer is formed, 400 nM of StrandDNA6 was addedto the cis chamber and mixed well. A control section was recorded for 5minutes at +180 mV to obtain DNA binding events to the nanopore. Afterthe control section was recorded, 800 nM of KF (exo minus) (NEB) wasadded and the signal was recorded for 5 minutes at +180 mV.

The open-pore level was visually estimated, and DNA translocation eventswere defined to be occasions when the data dropped below a thresholdplaced at about 5 sigma below the pore level (where sigma is thestandard deviation of the noise). Any obvious artifacts were manuallyremoved from the data before event detection was performed. Shown in thefigures is the mean current level of each event in pA (vertical axis) vsthe length of the event in seconds (horizontal axis). Note that thehorizontal axis is displayed using a logarithmic scale, since the eventlengths range from less than a millisecond to as much as 10 seconds. Inall four cases there were also numerous very short events (less than 1ms) which have been excluded. This is because they are too short fortheir current levels to be reliably estimated, and because they do notserve to distinguish between the different conditions shown.

StrandDNA1: 5′-Biotin- (SEQ ID NO: 22)TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGGCTACGACCTGCATGAGAATGC-3′ StrandDNA2: 5′-(SEQ ID NO: 23) CTCACCTATCCTTCCACTCACCCCCCAAAAAACCCCCCAAAAAACCCCCCAAAAAAGCA TTCTCATGCAGGTCGTAGCC-3′StrandDNA3: StrandDNA1 hybridised to StrandDNA2 (SEQ ID NO's: 22 and 23) StrandDNA4: 5′- (SEQ ID NO: 24)AACCCCCAAAAACCCCCAAAAACCCCCAAAAACCCCCAAAAACCCCCAAAAACCCCCAA AAACCCCCAAAAACCCCCATAGAGACAAGAATAACGAAGT A-3′ StrandDNA5:(SEQ ID NO: 25) 5′-TACTTCGTTATTCTTGTCTCTAT-3StrandDNA6: StrandDNA4 hybridised to StrandDNA5 (SEQ ID NO's: 24 and 25)2.2 Results

An experiment has been devised that allows a DNA handling protein to beassessed for its ability to hold onto DNA under the application of apotential. In this experiment, a DNA-enzyme complex is pulled into thenanopore resulting in a characteristic current level. When theDNA-enzyme complex dissociates, the DNA is pulled deeper into thenanopore resulting in a second current level. The DNA then completelytranslocates through the nanopore, resulting in an open pore andresetting the system to its original state. The kinetics of theDNA-enzyme binding can be assessed by examining the duration of theenzyme-bound state over multiple repeats of this process (FIG. 3).

In recent work, a polymerase has been used to control the translocationof a DNA strand. To run such an experiment, the DNA concentration isideally 100-1000 nM to be captured by the nanopore. As the enzyme bindsto the DNA, it is preferable for the enzyme concentration to be at asimilar molarity to the DNA, or in excess to the DNA. It is common forenzyme concentrations to be used at double the DNA concentration toensure that a large proportion (preferably all) of the DNA forms anenzyme-DNA complex. This places a high demand on the quantity ofmaterial required. It is therefore desirable be have a system that usesless DNA and hence, less enzyme.

One method of achieving this is to tether the DNA to the lipid membrane.As presented, the rate of DNA insertion can be greatly increased byenhancing the interaction between DNA and membrane. This can producerates that are comparable to those when the DNA is free in solution, butusing 1,000 to 10,000 times less material. By using a lowerconcentration of DNA, the concentration of enzyme used can be greatlyreduced (see FIG. 4).

A suitable DNA handling protein is the Klenow Fragment (KF) (N. A.Wilson, R. Abu-Schmays, B. Gyarfas, H. Wang, K. R. Lieberman, M. Akesonand W. B. Dunbar (2009). Electronic Control of DNA Polymerase Bindingand Unbinding to Single DNA Molecules. ACS Nano 3, 995-1003). The Klenowfragment is a large protein fragment produced when DNA polymerase I fromE. coli is enzymatically cleaved by the protease subtilisin. It retainsthe 5′-3′ polymerase activity and the 3′→5′ exonuclease activity forremoval of precoding nucleotides and proofreading, but loses its 5′→3′exonuclease activity. The KF can also be genetically engineered toremove the remaining 3′→5′ exonuclease activity. This DNA handlingprotein typically binds to the DNA at the interface between singlestranded and double stranded DNA (primer/template junction) and cancatalyse the replication of the DNA strand through the addition ofnucleotides. Klenow fragment has been investigated for Strand Sequencingapproaches but has been found to have binding times of 1-100 ms whenpulled on top of a nanopore by the application of a potential.

We screened the KF in a membrane tethered analyte setup as shown above(FIG. 4). When the DNA is in solution, the binding time of the KF-DNAcomplex is 1-100 ms (FIGS. 5 and 6) (similar to published results (refWilson/Akeson 2009)). This is too short to be useful for a StrandSequencing method as a duration of 100 ms would only allow a fewnucleotides to be read. However, when the DNA is tethered to the lipidmembrane, the binding time increases to 0.1-10 s (FIGS. 7 and 8).

2.3 Conclusions

The duration of the enzyme-DNA complex on top of the pore is a functionof the force from the applied field acting on the charged DNA strand.The ability of the protein to resist this force determines the length oftime that the complex remains intact on top of the pore. The longerdwell time for the tethered DNA may be due to the mobile lipid moleculesapplying an additional force on the strand in the pore as it diffusesacross the lipid membrane. This force negates the force applied by theapplied field and the net force that the KF experiences is reduced. Thissetup benefits from the advantages that a high field offers (e.g. highersignal to noise, faster DNA capture), but still allows the DNA handlingprotein to have a long binding time on top of the pore.

The tethering approach offers another means for controlling enzymebehaviour on top of the nanopore. There are many possibilities forexploring this concept. By varying the composition of the membrane, orchanging a physical parameter, such as temperature, it would be possibleto change the diffusion rate of the tethered molecule in the lipidbilayer, and hence, the force that the DNA-enzyme complex experiences atthe nanopore. In the embodiment of exonuclease sequencing, increasingmembrane fluidity may increase availability of polynucleotide toexonuclease. Membrane fluidity can be changed by adding agents such ascholesterol. In addition, the nature of the tethering agent could bechanged to control the diffusion rate of the tethered analyte to producea similar effect. It is likely that tethering to a large species, suchas a protein would yield a slower diffusion rate compared to tetheringto a small molecules such as a lipid. It has been shown that the enzymerate when it is complexed with polynucleotide and drawn into thenanopore will be affected by the field applied across the nanopore. Itis likely that the diffusional force from analyte tethering will reducethe net force that the enzyme on the pore experiences. We anticipatebeing able to control the rate of polymer movement by combining theforce from an applied potential with the diffusional force from analytetethering. Another potential use of this effect is to control the strandspeed through the pore without the use of a DNA handling protein. Theforce applied by the applied potential could be matched by thediffusional force of the membrane.

3. Example 3—More Strand Sequencing

In recent work, Phi29 DNA polymerase has been used to control thetranslocation of a DNA strand through α-hemolysin (Akeson et al., 2010,J Am Chem. Soc. 2010 Dec. 22; 132(50):17961-72.). Two modes ofcontrolled movement of a DNA strand through a nanopore have beenreported using Phi29 as a molecular motor, both methods relying on itsaction at the double/single stranded DNA juncture on a 5′-overhangingduplex. Movement can occur either by polymerisation from the primingstrand that is hybridised opposite to the strand being interrogated orby an unzipping method where the priming strand is sequentiallyunhybridised from the strand being interrogated to reveal more and moreof the targets sequence that was previously duplex DNA.

3.1 Materials and Methods

As presented for single stranded DNA, the tethering moiety can be variedto generate strands that display either a transient interaction with thebilayer or a more long duration tethering, for example with cholesterolof biotin:streptavidin respectively. For dsDNA analytes it might beconsidered that duplex DNA analytes which display transient bindingbehaviour might be more suitable for strand sequencing so as to enablethe enzyme to fully unzip the analyte and clear the nanopore ready forthe next.

Complementary Oligos (ONLA1346 and ONLA1347, 65 nt and 31 ntrespectively) were designed that contained on the target strand(ONLA1346) a cholesterol group at the 3′ and a polyC extensioncontaining a single A at the 5′. When hybridised these Oligos give a DNAduplex of 31 bp with a 34 nt 5′ overhang so that the target strand canbe threaded into and captured by the nanopore 5′ first. The unzippingcan then be tracked by looking at the movement of the single A, in thepolyC background, through the reader head of the nanopore. Forcomparison with non-tethered analytes a strand identical in sequence tothe target strand was designed but which lacked the cholesterol group(ONLA1049).

Single channel recordings were performed using a mutant MspA-NNNRRK pore(ONLP2726) in combination with Phi29 DNA polymerase. A single channelwas obtained and the cis buffer perfused with 10 ml of fresh buffer (400mM KCl, 10 mM HEPES pH8.0) to minimise the chance of single channelinsertion. After a 5 minute control section DNA was added to either 0.5nM or 100 nM, for tethered and non-tethered experiments respectively. Anumber of short duration events (˜10 ms) were observed after theaddition of DNA that are proposed to be the duplex DNA being captured bythe nanopore and the primer being stripped from the template by theforce of the pore. After 5 mins Phi29 DNA polymerase was added to thecis chamber to give either 10 nM or 200 nM, again respectively fortethered and non-tethered experiments.

Oligonucleotides used:

ONLA1346 (SEQ ID NO: 26)CCCCCCCCCCCCCCCACCCCCCCCCCCCCCCCCCCTATTCTGTTTATGTT TCTTGTTTGTTAGCC-CholONLA1347 (SEQ ID NO: 27) GGCTAACAAACAAGAAACATAAACAGAATAG ONLA1049(SEQ ID NO: 28) CCCCCCCCCCCCCCCACCCCCCCCCCCCCCCCCCCTATTCTGTTTATGTTTCTTGTTTGTTAGCC PolyT-50mer_XXXX_Sense (SEQ ID NO: 29)TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTXXXXGGTTGTTTCTGTTGGTGCTGATATTGC PhiX 235 bp Antisense (SEQ ID NO: 30)Chol-GTTAGACCAAACCATGAAACCAACATA  PhiX 400 bp Antisense (SEQ ID NO: 31)Chol-GACCGCCTCCAAACAATTTAGACA PhiX 822 bp Antisense (SEQ ID NO: 32)Chol-GGCAATCTCTTTCTGATTGTCCAG3.2 Results

After addition of the Phi29 DNA polymerase a number of long durationevents were observed in both experiments that are proposed to be captureof the DNA:protein complexes. These events show a short dwell time at aconstant level before oscillating between states, which are thought tooccur on initiation of unzipping and the A beginning to move through thereader head of the pore. An example of this is shown in FIGS. 12 and 11for both the tethered (FIG. 9) and non-tethered (FIG. 10) experiments.

Analysis of all unzipping events for both the tethered and solution DNAevents show a broadly constituent pattern for the number of observedstates, the mean vs dwell time for each state and the mean vs standarddeviation for each (FIG. 11 for non-tethered and FIG. 12 for tethered).

The position of the cholesterol is not set as being at the 3′ of thetarget strand and can be varied to the 5′ of either the template orprimer strand or within a hairpin. Due to the requirement for the enzymeto sit at 3′ of the primer strand, the juncture between single anddouble stranded DNA it is thought this is not a suitable site fortethering, however this has not been demonstrated experimentally.

Whilst the tethering method works well for synthetic strands, where theattachment chemistry can be incorporated during the chemical synthesisof the oligonucleotide, applying it to samples derived from genomic DNAis more challenging. A common technique for the amplification ofsections genomic DNA is using polymerase chain reaction. Here using twosynthetic oligonucleotide primers a number of copies of the same sectionof DNA can be generated, where for each copy the 5′ of each strand inthe duplex will be the synthetic oligo. By using an antisense primerthat has a 5′ cholesterol group each copy of the target DNA amplifiedwill contain a cholesterol group for tethering. The only problem withanalyte generated by PCR is that it is either blunt ended or contains asingle 3′-A overhang, neither of which are suitable for threading into ananopore for strand sequencing. Addition of sections of single strandedDNA to the 5′ of duplex DNA is not easily possible. A chemical orenzymatic ligation can be done but neither are highly efficient and alsorequire further downstream reactions and purification steps. A PCRmethod was developed using a sense primer that as is usual contained acomplementary section to the start of the target region of genomic DNAbut was additionally proceeded with a 50 PolyT section. To prevent thepolymerase from extending the complementary strand opposite the polyTsection, to create a blunt ended PCR product as is normal, four abasicsites were added between the PolyT section and the complementary primingsection. These abasic sites will prevent the polymerase from extendbeyond this region and so the polyT section will remain as 5′ singlestranded DNA on each of the amplified copies (FIGS. 13 and 14).

Whilst this PCR method is an efficient way of attaching the 5′ leaderpolyT section, other methods for incorporating the attachment chemistryare possible however, such as using terminal transferase to add to the3′ or via T4 polynucleotide kinase and ATPγS to add a reactive thiol tothe 5′ for chemical coupling. However, this method allows generation oftethered analytes in a form suitable for strand sequencing where theonly limitation on size, and as such read length, is that imposed by thePCR (˜20 kb).

Single channel recordings were carried out as described above but usingthese genomic DNA amplified fragments in order to observe any unzippingevents (FIGS. 15 and 16). Several unzipping events were observed thatprogressed and also then exited of their own accord, so suggestingcomplete unzipping of the duplex DNA.

In order to observe an acceptable event rate for capturing DNA:proteincomplexes for strand sequencing from solution then 100 nM DNA and 200 nMPhi29 DNA polymerase is required. For the 800 bp fragment this isequivalent to ˜50 ug of dsDNA per experiment, assuming the 1 ml chambervolume as used above. Using tethered dsDNA analytes the same acceptableevent rate can be satisfied and exceeded using 0.1 nM DNA and 10 nMPhi29 DNA polymerase. For the 800 bp fragment this is equivalent to ˜50ng of dsDNA per experiment, assuming the 1 ml chamber volume as usedabove.

4. Example 4—Solid State Sequencing

The advantages demonstrated above for tethering to a lipid membrane canalso be extended to solid state nanopore experiments. Nanopores can beproduced in solids state materials and utilised in a similar manner tobiological nanopores. Their use and fabrication has been well documentedelsewhere (WO 00/79257; WO 00/78668; Dekker C, Nat. Nanotechnol. 2007April; 2(4):209-15; and Schloss J A, et al., Nat. Biotechnol. 2008October; 26(10):1146-53).

Nanopores in solid state materials, such as silicon nitride offeradvantages over the biological channels as the pores. Solid statematerials are far less fragile than lipid membranes. Nanopores in solidstate material can be formed in a factory and have a long shelf life,unlike biological membranes which are often formed in situ. Recentadvances with solid state nanopores also allow very thin materials suchas graphene to be used which have unique properties (Golovchenko J, etal., Nature. 2010 Sep. 9; 467(7312):190-3; Drndié M, et al., Nano Lett.2010 Aug. 11; 10(8):2915-21; and Dekker C, et al., Nano Lett. 2010 Aug.11; 10(8):3163-7). Nanogaps in graphene have also been proposed (Postma,2008, Rapid Sequencing of Individual DNA Molecules in GrapheneNanogaps).

A further embodiment of solid state membranes is to use a tunnelingcurrent between two or more electrodes embedded in the nanopore. As ananalyte passes through the pore (driven by a trans membrane potential),the analyte facilitates a tunneling current between electrode. Thiscurrent can be used to detect the identity of the analyte (Schlosssupra; U.S. Pat. No. 7,253,434; and WO 2008/092760).

An alternative method to nanopores is to use nanogaps in solid statematerials as sensors (Chen et al., Materials Today, 2010, 13(11):28-41).

Solid state nanopore experiments can benefit from the advantagesdescribed above for lipid membranes. A key difference between the twomembrane types is that amphiphilic membranes often are naturally mobile,essentially acting as a two dimensional fluid with lipid diffusion ratesof ˜10⁻⁸ cm s⁻¹, while membranes in materials like silicon nitride aresolid. Although there may be advantages to tethering an analyte to asurface in a static fashion, it is desirable for the analyte to be ableto move across with membrane so that multiple analyte molecules caninteract with the detector.

There are a number of schemes that could be employed to tether ananalyte to a solid state membrane (FIG. 17). The first approach would beto rely on the natural interaction of the analyte with an unmodifiedmembrane, such as Si₃N₄. However, this provides very little control overthe diffusion rate of the analyte on the surface. It is thereforepreferable to modify the surface, the analyte, or both the surface andthe analyte to provide the desired interaction.

Methods for chemically modifying solid state materials are well known inthe art. Solid state nanopores have also been chemically modified,either through self-assembly in solution or by driving the reactivespecies through the nanopore under an applied potential (WO2009/020682).

The first two schemes use a chemically modified membrane to produce asurface where the analyte can transiently interact with the layer (FIGS.17A, B).

In the first scheme, the tethering group of the analyte embeds itselfinto the modified layer (FIG. 17A). A long chain alkane could beattached to the surface and a tethering group such as cholesterol or analkane would be used. The surface modification could be achieved byusing a chloro-hexadecyl-dimethylsilane (or similar) and the methodsdescribed in WO 2009/020682.

In the second scheme, the tethering analyte does not embed into thelayer, but resides on the surface. This could be achieved usinghydrophobic as in the first scheme. In addition, similar methods couldalso be envisaged where the binding of the analyte to the surface ismediated by electrostatic, hydrogen bonding or Van der Waalsinteractions. The third scheme is the most similar to the membranes usedwith protein nanopores. In this embodiment, the solid state membrane ismodified to support a lipid monolayer (FIG. 17C). This approach has allthe benefits of the examples presented above for lipid membranetethering. Tethering can be achieved by using a cholesterol anchor orattaching, via the lipid headgroups, or through a receptor in themembrane. Methods for forming bilayers or monolayers on solid surfacesare well known in the art (Duran R S, et al., Langmuir. 2007 Mar. 13;23(6):2924-7; and Cremer P S, et al., Surface Science Reports. 2006;61:429-444). When the surface is made hydrophobic, a lipid monolayer canbe formed spontaneously from lipid vesicles in solution. The surface canbe made hydrophobic in a number of ways, including plasma treatments(such as CH₄) or chemical methods, such as chloro-silane chemistry (WO2009/020682), and gold-thiol coupling (Duran supra; and Cremer supra).

A fourth scheme for tethering analytes to membranes is to use a solidstate membrane as a support for a lipid bilayer (FIG. 17D). In thisembodiment, the detector element is the nanopore in the solid statemembrane. This approach has all the benefits of the examples presentedabove for lipid membrane tethering. If the surface is renderedhydrophilic, lipid bilayers will self assemble on the surface—an effectwhich is common for bilayers formed on glass surfaces (Cremer supra).For all the examples above, the solid state nanopore can be combinedwith a polynucleotide binding protein to form the detector.

Example 5

This Example describes how helicase-controlled DNA movement was notobserved for non-tethered DNA when exposed to an MspA nanopore embeddedin a tri-block co-polymer. The chip has 128 wells with platinumelectrodes and an aperture of 30 μm with a platinum common electrodeattached to the cap.

The monolayers were formed with a solution mixture of 50 mg/ml tri-blockco-polymer (TBCP 6-33-6, OH-PMOXA-(PEG linker)-PDMS-(PEGLinker)-PMOXA-OH, Polymer Source Product ID: P3691B-MOXZDMSMOXZ) in oil.The nanopore(MS-(G75S/G77S/L88N/D90N/D91N/D93N/D118R/Q126R/D134R/E139K)8) was thenadded to the chip in the buffer. Reagents were only added across the topof the chip (cis side) once the chip was formed.

The experiment were carried out with 625 mM sodium chloride, 25 mMpotassium ferricyanide, 75 mM potassium ferrocyanide, 100 mM HEPES, pH8.0 (buffer 1). The MspA mutant used wasMS-(G75S/G77S/L88N/D90N/D91N/D93N/D118R/Q126R/D134R/E139K)8. The DNAsequence used in this experiment was a double-stranded 400mer strand(SEQ ID NO: 39 shows the sequence of the sense strand).

SEQ ID NO: 39- TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGGTTGTTTCTGTTGGTGCTGATATTGCGCTCCACTAAAGGGCCGATTGACCCGGTGGTACCTTGGTTGTTTCTGTTGGTGCTGATATTGCTTTTGATGCCGACCCTAAATTTTTTGCCTGTTTGGTTCGCTTTGAGTCTTCTTCGGTTCCGACTACCCTCCCGACTGCCTATGATGTTTATCCTTTGGATGGTCGCCATGATGGTGGTTATTATACCGTCAAGGACTGTGTGACTATTGACGTCCTTCCCCGTACGCCGGGCAATAATGTTTATGTTGGTTTCATGGTTTGGTCTAACTTTACCGCTACTAAATGCCGCGGATTGGTTTCGCTGAATCAGGTTATTAAAGAGATTATTTGTCTCCAGCCACTTAAGTGAGGTGATTTATGTTTGGTGCTATTGCTGGCGGTATTGCTTCTGCTCTTGCTGGTGGCGCCATGTCTAAATTG TTTGGAGGCGGTCGAGCT

The monolayer was formed with 50 mg/ml tri-block co-polymer (TBCP6-33-6, OH-PMOXA-(PEG linker)-PDMS-(PEG Linker)-PMOXA-OH, Polymer SourceProduct ID: P3691B-MOXZDMSMOXZ) in oil and nanopores(MS-(G75S/G77S/L88N/D90N/D91N/D93N/D118R/Q126R/D134R/E139K)8)pre-inserted on the chip. The chip was then inserted into the blade andthe solution manually removed by pipette and re-inserted. Next 1.5 nMDNA (sense strand sequence SEQ ID NO: 39), 500 nM helicase, 10 mM MgCl₂and 1 mM ATP was added to 150 ul of buffer 1. The solution was thenpipetted across the chip through the chimney in the cap and left todiffuse to the nanopore. Data was recorded for 1 hour at +120 mV, with apotential flip to 0 mV and then −50 mV every 5 minutes, to obtainhelicase events in the nanopore.

Helicase-controlled DNA movement for non-tethered DNA (sense strandsequence SEQ ID NO: 39) through aMS-(G75S/G77S/L88N/D90N/D91N/D93N/D118R/Q126R/D134R/E139K)8 nanoporeinserted in a tri-block co-polymer (TBCP 6-33-6, OH-PMOXA-(PEGlinker)-PDMS-(PEG Linker)-PMOXA-OH, Polymer Source Product ID:P3691B-MOXZDMSMOXZ) was not detected. The pore was observed to blockunder the conditions tested but no helicase-controlled DNA movement wasnoted.

Example 6

This Example describes how helicase-controlled DNA movement was observedfor tethered DNA when exposed to an MspA nanopore embedded in atri-block co-polymer. The chip has 128 wells with platinum electrodesand an aperture of 30 μm with a platinum common electrode attached tothe cap.

The monolayers were formed with a solution mixture of 50 mg/ml tri-blockco-polymer (TBCP 6-33-6, OH-PMOXA-(PEG linker)-PDMS-(PEGLinker)-PMOXA-OH, Polymer Source Product ID: P3691B-MOXZDMSMOXZ) in oil.The nanopore(MS-(G75S/G77S/L88N/D90N/D91N/D93N/D118R/Q126R/D134R/E139K)8) was thenadded to the chip in the buffer. Reagents were only added across the topof the chip (cis side) once the chip was formed.

The experiment were carried out with 625 mM sodium chloride, 25 mMpotassium ferricyanide, 75 mM potassium ferrocyanide, 100 mM HEPES, pH8.0 (buffer 1). The MspA mutant used wasMS-(G75S/G77S/L88N/D90N/D91N/D93N/D118R/Q126R/D134R/E139K)8. The DNAsequence used in this experiment consists of double-stranded 400mer DNA(SEQ ID NO: 40 shows the sequence of the sense strand) and a shortcomplementary strand of DNA with a cholesterol attached at the 3′ end(SEQ ID NO: 41) which can hybridise to a portion of SEQ ID NO: 40.

SEQ ID NO: 40 TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGGTTGTTTCTGTTGGTGCTGATATTGCGCTCCACTAAAGGGCCGATTGACGCTCCACTAAAGGGCCGATTGACCCGGTTGTTTCTGTTGGTGCTGATATTGCTTTTGATGCCGACCCTAAATTTTTTGCCTGTTTGGTTCGCTTTGAGTCTTCTTCGGTTCCGACTACCCTCCCGACTGCCTATGATGTTTATCCTTTGGATGGTCGCCATGATGGTGGTTATTATACCGTCAAGGACTGTGTGACTATTGACGTCCTTCCCCGTACGCCGGGCAATAATGTTTATGTTGGTTTCATGGTTTGGTCTAACTTTACCGCTACTAAATGCCGCGGATTGGTTTCGCTGAATCAGGTTATTAAAGAGATTATTTGTCTCCAGCCACTTAAGTGAGGTGATTTATGTTTGGTGCTATTGCTGGCGGTATTGCTTCTGCTCTTGCTGGTGGCGCCATGTCTAAATTGTTTGGAGGCGGTCGAGCT SEQ ID NO: 41AGCGACTAACAAACACAATCTGATGGCTTTTTTTTTTTTTTTTTTTTTTT TTTTTTT/3 CholTEG/

The monolayer was formed with 50 mg/ml tri-block co-polymer (TBCP6-33-6, OH-PMOXA-(PEG linker)-PDMS-(PEG Linker)-PMOXA-OH, Polymer SourceProduct ID: P3691B-MOXZDMSMOXZ) in oil and nanopores pre-inserted on thechip. The chip was then inserted into the blade and the solutionmanually removed by pipette and re-inserted. Next 1.5 nM DNA (sensestrand sequence of SEQ ID NO: 40 and short complementary tether strandSEQ ID NO: 41), 500 nM helicase, 10 mM MgCl₂ and 1 mM ATP was added to150 ul of buffer 1. The solution was then pipetted across the chipthrough the chimney in the cap and left to diffuse to the nanopore. Datawas recorded for 1 hour at +120 mV, with a potential flip to 0 mV andthen −50 mV every 5 minutes, to obtain helicase controlled DNA movementthrough the nanopore.

Helicase-controlled translocation of tethered DNA (sense strand sequenceof SEQ ID NO: 40 and short complementary tether strand SEQ ID NO: 41)through a MS-(G75S/G77S/L88N/D90N/D91N/D93N/D118R/Q126R/D134R/E139K)8nanopore inserted in a tri-block co-polymer (TBCP 6-33-6, OH-PMOXA-(PEGlinker)-PDMS-(PEG Linker)-PMOXA-OH, Polymer Source Product ID:P3691B-MOXZDMSMOXZ) was detected. Twelve helicase-controlled DNAmovements were detected during the course of 1, 5 minute positive cycle.The median time between helicase-controlled DNA movements was 0.5seconds. Therefore, by tethering the DNA to the tri-block co-polymer itis possible to observe helicase-controlled DNA movement which was notdetected in a similar experiment using non-tethered DNA (example 5).

The invention claimed is:
 1. A method for detecting polynucleotides,comprising: (a) providing a membrane in which is present a nanopore thatprovides a channel through the membrane; (b) contacting the membrane, inan ionic solution, with polynucleotides, wherein following contact withthe membrane the polynucleotides are tethered to the membrane via atethering group; (c) applying a potential difference across the membraneand detecting a first polynucleotide using the nanopore, from among thepolynucleotides tethered to the membrane; and (d) detecting a secondpolynucleotide, from among the polynucleotides tethered to the membrane,using the same nanopore, wherein the second polynucleotide is not thefirst polynucleotide.
 2. The method according to claim 1, wherein themembrane is an amphiphilic layer or a solid state layer.
 3. The methodaccording to claim 1, wherein the membrane is a lipid bilayer.
 4. Themethod according to claim 1, wherein the tethering group comprises ahydrophobic anchor.
 5. The method according to claim 4, wherein thehydrophobic anchor is a lipid, fatty acid, sterol, carbon nanotube oramino acid.
 6. The method according to claim 4, wherein the hydrophobicanchor is capable of embedding in the membrane.
 7. The method accordingto claim 1, wherein the tethering group comprises a linker.
 8. Themethod according to claim 7, wherein the linker comprises apolynucleotide, polyethylene glycol, polysaccharide, or polypeptide. 9.The method according to claim 1, wherein the polynucleotide is tetheredtransiently to the membrane.
 10. The method according to claim 1,wherein the first polynucleotide and the second polynucleotide aredetected based on ion flow through the nanopore that is measured via anelectrical means.
 11. The method according to claim 1, wherein thenanopore is a protein nanopore.
 12. The method according to claim 11,wherein the protein nanopore is derived from Msp or α-hemolysin (α-HL).13. The method according to claim 1, wherein the nanopore comprises amolecular adaptor that mediates interaction of the polynucleotide withthe nanopore.
 14. The method according to claim 1, wherein the nanoporeis coupled to a polynucleotide binding protein, which is optionally anexonuclease or a polymerase.
 15. The method according to claim 1,wherein the first polynucleotide and the second polynucleotide aredetected based on ion flow through the nanopore that is measured bymeasuring a current passing through the nanopore.
 16. The methodaccording to claim 1, wherein the polynucleotide comprises a targetpolynucleotide.
 17. The method according to claim 16, wherein the methodcomprises digesting a target polynucleotide to provide a fragment andthe fragment sequence is determined.
 18. The method according to claim1, wherein the tethering group comprises a polypeptide.
 19. The methodaccording to claim 1, wherein the polynucleotide is present in thesolution of (b) at a concentration of less than 0.01 pM.
 20. The methodaccording to claim 1, wherein the polynucleotide is present in thesolution of (b) at a concentration of less than 0.1 pM.
 21. The methodaccording to claim 1, wherein the polynucleotide is present in thesolution of (b) at a concentration of less than 1 pM.
 22. The methodaccording to claim 1, wherein the polynucleotide is present in thesolution of (b) at a concentration of less than 10 pM.
 23. The methodaccording to claim 1, wherein the polynucleotide is present in thesolution of (b) at a concentration of less than 100 pM.
 24. The methodaccording to claim 1, wherein the polynucleotide is present in thesolution of (b) at a concentration of about 0.001 pM to about 1 nM. 25.The method according to claim 1, wherein rate of interaction of themolecules of the polynucleotide with the nanopore is increased ascompared to rate of interaction of molecules of the polynucleotide withthe nanopore in the absence of the tethering group.
 26. The methodaccording to claim 25, wherein the rate of interaction of the moleculesof the polynucleotide is increased by at least three orders ofmagnitude.
 27. The method according to claim 1, wherein the effectiveconcentration of the polynucleotide at the nanopore is increased ascompared to the concentration of the polynucleotide in the solution of(b).
 28. The method according to claim 1, wherein a single nanoporeproviding an ion channel through the membrane is present in themembrane.
 29. The method according to claim 1, wherein thepolynucleotide is functionalized with the tethering group.